Wnt pathway stimulation in reprogramming somatic cells with nuclear reprogramming factors

ABSTRACT

The invention provides compositions and methods of use in reprogramming somatic cells. Compositions and methods of the invention are of use, e.g., for generating or modulating (e.g., enhancing) generation of induced pluripotent stem cells by reprogramming somatic cells. The reprogrammed somatic cells are useful for a number of purposes, including treating or preventing a medical condition in an individual. The invention further provides methods for identifying an agent that reprograms somatic cells to a pluripotent state and/or enhances the speed and/or efficiency of reprogramming. Certain of the compositions and methods relate to modulating the Wnt pathway.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application 60/967,028, filed Aug. 31, 2007, and U.S. ProvisionalPatent Application 61/188,190, filed Aug. 6, 2008, both of which areincorporated herein by reference.

The invention described herein was supported, in whole or in pert, bygrants 5-ROI-HDO45022, 5-R37-CA084198, and 5-ROI-CA067869 to RJ and byNIH grant HG002668 from the National Institutes of Health. The UntiedStates government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Stem cells are cells that are capable of self-renewal and of giving riseto more differentiated cells. Embryonic stem (ES) cells candifferentiate into the multiple specialized cell types that collectivelycomprise the body. In addition to being of immense scientific interest,the property of pluripotency gives human ES cells great clinical promisefor applications m regenerative medicine such as cell/tissue replacementtherapies for disease.

Several different methods are currently used to obtain ES cells. In onemethod, an ES cell line is derived from the inner cell mass of a normalembryo in the blastocyst stage (See U.S. Pat. Nos. 3,843,780 and6,200,806, Thompson, J. A. et al. Science, 282:1143-7, 1998). A secondmethod for creating pluripotent ES cells utilizes somatic cell nucleartransfer (SCNT). In this technique, the nucleus is removed from a normalegg, thus removing the genetic material. The nucleus of a donor diploidsomatic cell is introduced directly into the enucleated oocyte, e.g., bymicromanipulation, or the donor diploid somatic cell is placed next tothe enucleated egg and the two cells are fused. The resulting cell hasthe potential to develop into an early embryo from which the portioncontaining the stem cell producing inner cell mess can be obtained. In athird method, the nucleus of a human cell is transplanted into anenucleated animal oocyte of a species different from the donor cell.See, e.g., U.S. Pat. Pub. No. 20010012513. The resultant chimeric cellsare used for the production of pluripotent ES cells, in particularhuman-like pluripotent ES cells. Disadvantages of this technique arethat these chimeric cells may contain unknown viruses and retain themitochondria of the animal species.

The traditional ES cell isolation methods suffer from severallimitations when applied to generating human ES cells. These includeethical controversies associated with the source of the cells as well astechnical challenges. A significant limitation to the productiveutilization of ES cells for clinical applications is the difficultyassociated with generating ES cells that are genetically matched toindividual patients. There exists a significant need for alternativemethods of generating pluripotent cells.

SUMMARY OP THE INVENTION

The present invention provides compositions and methods forreprogramming somatic cells to a less differentiated state. In certainembodiments the compositions and methods permit reprogramming of somaticcells to pluripotent, embryonic stem cell-like cells (“ES-like cells”).

In one aspect, the invention provides a method of reprogramming asomatic mammalian cell comprising culturing the cell in the presence ofan extracellular signaling molecule so that the cell becomesreprogrammed.

In one aspect the invention provides a method of reprogramming a somaticmammalian cell comprising culturing the cell in Wnt conditioned cellculture medium so that the cell becomes reprogrammed. In certainembodiments the method comprises culturing the somatic cell so that thecell is induced to become pluripotent. In certain embodiments the Wntconditioned cell culture medium comprises Wnt3a conditioned medium(Wnt3a-CM).

In another aspect the invention provider a method of reprogramming asomatic mammalian cell comprising contacting the cell with an agent thatincreases the activity of a Wnt pathway so that the cell is induced tobecome pluripotent. In some embodiments the agent is a soluble,biologically active Wnt protein, e.g., a Wnt3a protein. In someembodiments the agent is selected from the group consisting of: (i)small molecules that mimic the effect of Wnt3a conditioned medium orsoluble, bio logically active Wnt proteins, e.g., by interacting withcell receptors) for Wnt; (ii) agents that modulate the interactionbetween β-catenin and a member of the TCF/LEF family and/or modulate theexpression or activity of a member of the TCF/LEF family, (iii) agentsthat inhibit expression or activity of an endogenous inhibitor of theWnt pathway.

The invention provides somatic cells reprogrammed using the inventivemethods.

Cell culture media containing a Wnt3a activator and an additionalreprogramming agent capable of substituting for engineered expression ofOct4, Klf4, and/or Sox2 (or any combination thereof) are additionalaspects of this invention. Further aspects of the invention are (I) acomposition comprising: (i) a cell that has been modified to increaseits expression of Oct4, Klf4, and/or Sox2, or any subset of these; and(ii) a Wnt pathway modulator, e.g., a Wnt pathway activator; (2) acomposition comprising: (i) a cell that has been modified to increaseits expression or intracellular level of one or more reprogrammingfactors, wherein the reprogramming factor(s) is/are optionally selectedfrom Oct4, Klf4, and/or Sox2, or any subset of these; and (ii) Wntconditioned medium; (3) a composition comprising: (i) a cell that hasbeen modified to increase its expression or intracellular level of oneor more reprogramming factors, wherein the reprogramming factor(s)is/are optionally selected from Oct4, Nanog, Lin28 and/or Sox2, or anysubset of these; and (ii) a Wnt pathway activator; and (4) a compositioncomprising: (i) a cell that has been modified to increase its expressionor intracellular level of one or more reprogramming factors, wherein thereprogramming factor(s) is/are optionally selected from Oct4, Nanog,Lin28 and/or Sox2, or any subset of these; and (ii) Wnt conditionedmedium.

The invention also provides methods for identifying an agent thatreprograms somatic cells to a less differentiated state and/orcontributes to such reprogramming in combination with one or more otheragents. In certain of the methods, somatic cells are contacted with anagent that increases Wnt pathway activity and a candidate agent. Cellsare assessed for pluripotency characteristics. The presence of it leasta subset of pluripotency characteristics indicates that the agent ascapable of reprogramming somatic cells to a less-differentiated state.The agents identified by the present invention can then by used toreprogram somatic cells by contacting somatic cells with the agents.

The present invention further provides methods for treating a conditionin an individual in need of treatment for a condition. In certainembodiments, somatic cells are obtained from the individual andreprogrammed by the methods of the invention. The reprogrammed cells maybe expanded in culture. Pluripotent reprogrammed cells (which refers tothe original reprogrammed cells and/or their progeny that retain theproperty of pluripotency) are maintained under conditions suitable forthe cells to develop into cells of a desired cell type or cell lineage.In some embodiments, the cells are differentiated m vitro usingprotocols, such as those known in the art. The reprogrammed cells of adesired cell type are introduced into the individual to treat thecondition. In certain embodiments, the somatic cells obtained from theindividual contain a mutation in one or more genes. In these instances,in certain embodiments the somatic cells obtained from the individualare first treated to repair or compensate for the defect, e.g., byintroducing one or more wild type copies of the gene(s) into the cellssuch that the resulting cells express the wild type version of the gene.The cells are then introduced into the individual.

In certain embodiments, the somatic cells obtained from the individualare engineered to express one or more genes following their removal fromthe individual. The cells may be engineered by introducing a gene orexpression cassette comprising a gone into the cells. The introducedgene may be one that is useful for purposes of identifying, selecting,and/or generating a reprogrammed cell. In certain embodiments theintroduced gene(s) contribute to initiating and/or maintaining thereprogrammed state, in certain embodiments the expression produces) ofthe introduced gene(s) contribute to producing the reprogrammed statebut are dispensable for maintaining the reprogrammed state.

In certain other embodiments, methods of the invention can be used totreat individuals in need of a functional organ. In the methods, somaticcells are obtained from an individual in need of a functional organ, andreprogrammed by the methods of the invention to produce reprogrammedsomatic cells. Such reprogrammed somatic cells are then cultured underconditions suitable for development of the reprogrammed somatic cellsinto e desired organ, which is then introduced into the individual.

In Anther summary, the invention provides a method of reprogramming asomatic mammalian cell comprising contacting the somatic mammalian cellwith an agent that modulates a Wnt pathway so that the somatic mammaliancell becomes reprogrammed, in certain embodiments of the invention themethod comprises reprogramming the somatic mammalian cell to apluripotent state. In certain aspects, the invention providesimprovements in methods of generating induced pluripotent stem (iPS)cells. For example, in certain aspects the invention enhancesreprogramming somatic cells to pluripotency that have not beenengineered to express c-Myc. In certain aspects, the inventive methodsfacilitate generating homogenous ES-like colonies. In some embodiments,the inventive methods enhance formation of homogenous, ES-like colonieswithout imposing a selection step that requires genetic modification ofthe initial somatic cells.

In certain embodiments of the invention, the method comprises culturingthe cell in Wnt-conditioned medium. In certain embodiments, the methodcomprises culturing the cell in Wnt3a-conditioned medium. In certainembodiments, the cell is a human cell. In certain embodiments the cellis a mouse cell. In certain embodiments, the cell is a non-human primatecell. In certain embodiments, the somatic mammalian cell is a terminallydifferentiated cell. In certain embodiments the cell is a fibroblast orimmune system cell (e.g., B or T cell). In certain embodiments, thesomatic mammalian cell is not a terminally differentiated cell. Forexample, the somatic mammalian cell may be a precursor cell, e.g., aneural precursor or hematopoietic precursor cell. In certainembodiments, the method is practiced in vitro. In certain embodiments,contacting the cell comprises culturing the cell in culture mediumcontaining the agent. In certain embodiments, contacting comprisesculturing the cell in culture medium comprising the agent for at leant10 days. In certain embodiments, contacting comprises culturing the cellin culture medium comprising the agent for at least 12 or at least 15days or at least 20 days. In certain embodiments, the somatic cell isgenetically modified to contain a nucleic add sequence encoding aselectable marker, operably linked to a promoter for an endogenouspluripotency gene thereby allowing selection of cells that have beenreprogrammed to pluripotency while in other embodiments the somatic cellis not genetically modified to contain a nucleic acid sequence encodinga selectable marker operably linked to a promoter for an endogenouspluripotency gene thereby allowing selection of cells that have beenreprogrammed to pluripotency. In certain embodiments, the somatic cellis modified to express or contain at least one reprogramming factor atlevels greater than normally present in somatic cells of that type. Insome embodiments, the reprogramming factor is Oct4. In some embodiments,the reprogramming factor is Sox2. In some embodiments the reprogrammingfooter is Klf4. In some embodiments the reprogramming factor is Nanog.In some embodiments the reprogramming factor is Lin28. In someembodiments the reprogramming factor(s) are Oct4 and Sox2, In someembodiments the reprogramming factor(s) are Oct4, Sox2, and Klf4. Incertain embodiments, the somatic cell is not genetically modified toexpress c-Myc at levels greater than normally present in somatic cellsof that cell type. In certain embodiments, the cell is also contactedwith a second agent that modulates the Wnt pathway. In certainembodiments, the somatic cell is cultured in medium containing exogenoussoluble, biologically active Wnt protein. In certain embodiments, theWnt protein is Wnt3a protein. In certain embodiments, the method forther comprises confirming that the reprogrammed cell is pluripotent. Incertain embodiments, the method is practiced on a population of cellsand the method further comprises separating cells that are reprogrammedto a pluripotent state from cells that are not reprogrammed to apluripotent state. In certain embodiments, the method further comprisesadministering the reprogrammed cell to a subject. In certainembodiments, the method further comprises differentiating the cell to adesired cell type in vitro after reprogramming the cell. In certainembodiments, the method further comprises administering thedifferentiated cell to a subject.

The invention also provides a method of treating an individual in needthereof comprising: (a) obtaining somatic cells from the individual; (b)reprogramming at least some of the somatic cells by a method comprisingcontacting the somatic mammalian cells with an agent that modulates theWnt pathway (e.g., a Wnt pathway activator); and (c) administering atleast some of the reprogrammed cells to the individual, optionally afterdifferentiating the cells into one or more desired cell types. In someembodiments, the individual is a human. In some embodiments, the methodis practiced on a population of cells and farther comprises separatingcells that are reprogrammed to a pi on potent state from cells that arenot reprogrammed to a pluripotent state. In some embodiments, the methodfarther comprises differentiating the cell m vitro and, optionally,administering the differentiated cell to an individual in need oftreatment for a condition for which cell therapy is of use. For example,cells may be differentiated along a desired cell lineage such as aneural lineage, a muscle lineage, etc.

The invention farther provides composition comprising (i) a somaticmammalian cell that has been modified or treated so that it expresses orcontains at least one reprogramming factor at levels greater than wouldbe the case without such modification or treatment; and (ii) an agentdial increases activity of a Wnt pathway and contributes toreprogramming the somatic cell to a pluripotent state. In certainembodiments, the agent is Wnt3a protein. In certain embodiments, theagent is a small molecule.

The invention farther provides a method of identifying an agent usefulfor modulating the reprogramming of mammalian somatic cells to apluripotent state comprising: (a) culturing a population of mammaliansomatic cells in medium containing an agent that modulates activity of aWnt pathway and a candidate agent; and (b) determining; after a suitableperiod of time, whether cells having one or more characteristics of EScells are present after maintaining the cells and their progeny inculture for a suitable lime period, wherein the candidate agent isidentified as being useful for modulating the reprogramming of mammaliansomatic cells to a pluripotent state. If cells having one or morecharacteristics of ES cells are present at levels different than wouldbe expected had the medium not contained the candidate agent.

In certain embodiments, the characteristics are selected from: colonymorphology, expression of an endogenous gene expressed selectively by EScells, expression of a detectable marker operably linked to expressioncontrol sequences of a gene expressed selectively by ES cells, abilityto differentiate into cells having characteristics of endoderm,mesoderm, and ectoderm when injected into immunocompromised mice, andability to participate in formation of chimeras that survive to term. Incertain embodiments, the cells have been modified to express at leastone reprogramming factor. In certain embodiments, the medium isWnt-conditioned medium.

In certain embodiments, the medium is Wnt3a-conditioned medium, incertain embodiments, the agent that modulates activity of a Wnt pathwayis Wnt3a protein, in certain embodiments, the agent that modulatesactivity of a Wnt pathway is a small molecule. In certain embodiments,the candidate agent is a small molecule. In certain embodiments, themethod comprises identifying an agent useful for enhancing thereprogramming of mammalian somatic cells, wherein the candidate agent isidentified as being useful for enhancing the reprogramming of mammaliansomatic cells to a pluripotent state if cells having one or morecharacteristics of ES cells are present at levels greater than would beexpected had the medium not contained the candidate agent. In certainembodiments, step (b) comprises determining whether cell colonies havingone or more characteristics of ES cell colonies are present aftermaintaining the cells and their progeny in culture for a suitable timeperiod, wherein the candidate agent is identified as being useful formodulating the reprogramming of mammalian somatic cells to a pluripotentstate if cell colonies having one or more characteristics of ES cellcolonies are present at levels different than would be expected had themedium not contained the candidate agent. In certain embodiments, thecells express at least one reprogramming factor.

The invention also provides a method of identifying an agent useful forreprogramming mammalian somatic cells to a pluripotent state comprising:(a) contacting a population of mammalian somatic cells with an agentthat increases Wnt pathway activity and a candidate agent; (b)maintaining the cells in a cell culture system for a suitable period oftime; and (c) determining whether cells having one or morecharacteristics of ES cells are present in said culture system, whereinthe agent is identified as being useful for reprogramming mammaliansomatic cells to an ES-like state if cells having one or morecharacteristics of ES cells are present at levels greater than would beexpected had the cells not been contacted with the candidate agent.

In certain embodiments of the invention, the characteristics areselected from: colony morphology, expression of an endogenous geneexpressed selectively by ES cells, expression of a detectable markeroperably linked to expression control sequences of a gene expressedselectively by ES cells, ability to differentiate into cells havingcharacteristics of endoderm, mesoderm, and ectoderm when injected intoimmunocompromised mice, and ability to participate in formation ofchimeras that survive to term.

In certain embodiments, the agent that increases Wnt pathway activity isWnt3a protein. In certain embodiments, the candidate agent is a smallmolecule. In certain embodiments, the cells express at least onereprogramming factor. In certain embodiments, step (b) comprisesdetermining whether cell colonies having one or more characteristics ofES cell colonies are present after maintaining the cells and theirprogeny in culture for a suitable time period, wherein the candidateagent is identified as being useful for modulating the reprogramming ofmammalian somatic cells to a pluripotent state if cell colonies havingone or more characteristics of ES cell colonies are present at levelsdifferent than would be expected had the medium not contained thecandidate agent.

The invention also provides a method of reprogramming a somaticmammalian cell comprising culturing the cell in the presence of anextracellular signaling molecule so that the cell becomes reprogrammed.In certain embodiments, said extracellular signaling molecule is amolecule whose binding to an extracellular domain of a cellular receptorinitiates or modifies a signal transduction pathway within the cell. Incertain embodiments, the signal transduction pathway is the Wnt pathway.

The invention also provides a method of identifying a Wnt pathwaymodulator useful formulating the reprogramming of mammalian somaticcells to a pluripotent state comprising: (a) culturing a population ofmammalian somatic cells in medium containing the Wnt pathway modulator,(b) determining, after a suitable period of time, whether cells havingone or more characteristics of ES cells are present after maintainingthe cells and their progeny in culture for a suitable time period,wherein the Wnt pathway modulator is identified as being useful formodulating the reprogramming of mammalian somatic cells to a pluripotentstate if cells having one or more characteristics of ES cells erepresent et levels different than would be expected had the medium notcontained the Wnt pathway modulator.

In certain embodiments, the method comprises (i) testing at least 10 Wntpathway modulators; and (ii) identifying one or more of the Wnt pathwaymodulators as having significantly greater effect on reprogramming speedor efficiency than at least 50% of the other Wnt pathway modulatorstested. In certain embodiments, the method comprises testing at least20, at least 50, or at least 100 Wnt pathway modulators. In someembodiments, the method comprises identifying one or more of the Wntpathway modulators as having significantly greater effect onreprogramming speed or efficiency than at least 75%, or at least 90% ofthe other Wnt pathway modulators tested. In certain embodiments, the Wntpathway modulators tested are small molecules. In certain embodiments,the Wnt pathway modulators tested are structurally related. For example,they may be members of a set of compounds, e.g., a combinatorialcompound library, synthesized based on a common core structure or theymay be derivatives obtained by modifying a core structure or leadcompound such as by making substitutions or additions at one or morepositions. In certain embodiments, the Wnt pathway modulator isidentified as being useful for increasing the speed or efficiency ofreprogramming cells to an ES-tike state if, after a suitable timeperiod, cells having one or more characteristics of ES cells ere presentin numbers greater than would be expected had the medium not containedthe Wnt pathway modulator. In certain embodiments, the Wnt pathwaymodulator is identified as being useful for increasing the speed orefficiency of reprogramming cells to a pluripotent state if, after asuitable time period, cell colonies having one or more characteristicsof ES cell colonies, are present in numbers greater than would beexpected had the medium not contained the Wnt pathway modulator. Forexample, the methods may result in an increased percentage of colonieshaving features of ES cell colonies and/or the colonies may be morehomogenous than would be the case in the absence of the Wnt pathwaymodulator.

The invention further provides a cell culture composition comprising:(a) cell culture medium containing a Wnt pathway modulator; and (b) aplurality of mammalian somatic cells, wherein (i) the cells aregenetically modified or transiently transfected to express one or morereprogramming factors; (ii) the cells are genetically modified tocontain a nucleic acid sequence encoding a selectable marker, operablylinked to a promoter for an endogenous pluripotency gene, therebyallowing select km of cells have been reprogrammed to pluripotency, or(iii) the cell culture medium contains one or more small molecules,nucleic acids, or polypeptides that substitute for a reprogramming Actorother than c-Myc.

In certain embodiments, the cell culture medium comprises Wnt-3a CM. Incertain embodiments, the medium contains a small molecule that modulatesthe Wnt pathway.

In certain embodiments, the one or more reprogramming Adore are selectedfrom: Oct4, Nanog, Sox2, Lin2l, and Klf4. The invention further providesa composition comprising: an iPS cell and an agent that modulates, e.g.,activates, the Wnt pathway. In certain embodiments the agent thatactivates the Wnt pathway is Wnt3a protein. In certain embodiments theagent that activates the Wnt pathway is a small molecule.

In certain embodiments, the invention provides use of an agent thatmodulates a Wnt pathway in the manufacture of a medicament forreprogramming a somatic mammalian cell.

It is contemplated that all embodiments described herein are applicableto the various aspects of the invention. It is also contemplated thatthe various embodiments of the invention and elements thereof can becombined with one or more other such embodiments and/or elementswhenever appropriate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Wnt3a promotes epigenetic reprogramming, a. Schematicrepresentation of the experimental time-line. MEFs were infected withDOX-inducible lentivirus, split into cultures with and without Wnt3-CMtreatment, and then induced with DOX (day 0). G418 selection wasinitialed at fixed time points after induction and Wnt3a-CM treatmentwas maintained for 7 days of selection. DOX and G418 were maintaineduntil resistant colonies were assessed, b. G418-resistant colony countsfrom MEFs overexpressing Oct4/Sox2/Klf4/c-Myc in standard ES cell mediaor with Wnt3a-CM treatment, c. Phase images of G418 resistant coloniesformed with and without Wnt3a-CM treatment, d. G418-resistant colonycounts from MEFs infected with different combination of reprogrammingfedora in the presence and absence of Wnt3a-CM. G418 resistant coloniesemerged without c-Myc transduction in the presence of Wnt3a-CM. e. Phaseimage of Myc[−] G418 resistant colony formed with Wnt3a-CM treatment. Inthis experiment, no colonies were observed for Myc[−] cells in theabsence of Wnt3a-CM. f. G418-resistant colony counts from MEFsover-expressing Oct4/Sox2/Klf4 (Myc[−]) or Oct4/Sox2/Klf4/c-Myc (Myc[+])in the presence (red bars) and absence (gray bars) of Wnt3a-CM. G418selection was initiated on day 5 or day 10 post-induction as indicatedand colonies (in a 32-cm² area) were assessed on day 20 μg. Scatterplots comparing GFP intensity to autofluorescence, using flow cytometry,in Oct4-GFP cells on day 20 post-induction of Oct4/Sox2/Klf4, reveal aGFP expressing population of cells (indicated with an arrow) only withWnt3a-CM treatment, h. Phase image of GFP expressing Myc[−] cellsderived with Wnt3a-CM treatment and without any genetic selection.

FIG. 2. Induction of Pluripotency in Wnt Stimulated cells, a-d.Immunostaining reveals induction of pluripotency markers, Nanog (a-b)and SSEA-1(c-d) in Wnt3a-CM treated Myc[−] cells, e-g. Wnt3a-CM treatedMyc[−] tines formed teratomas when injected into SOD micesubcutaneously. Teratomas from Oct4/Sox2/Klf4/Wnt3aCM iPS tines showedevidence of differentiated cells of three germ layers similar toteratomas formed from V6.5 mES injections. Arrows indicated neuraltissue in (e), cartilage in (f), and endodermal cells in (g), h.Oct4/Sox2/Klf4/Wnt3aCM iPS lines derived without selection gave rise tochimeric mice (as shown on the left) with agouti coat color andpigmented eyes (in contrast to wild type Balb/c mouse, right) providingevidence of contribution so somatic cells. Coat color of offspringconfirmed that the Oct4/Sox2/Klf1/Wnt3aCM iPS line generated here isgermline-competent (data not shown).

FIG. 3. Wnt/β-catenin stimulation enhances iPS colony formation inabsence of c-Myc retrovirus, a. Counts are shown for G418 resistantcolonies in Oct4/Sox2/Klf4 over-expressing MEFs cultured in ES cellmedia, MEF conditioned media, Wnt3a over-expressing conditioned media,and Wnt3a over-expressing conditioned media with ICG001 (4 μM).Selection was initiated on day 15 post-induction, and colonies wereassessed on day 28. Wnt3a-CM treatment was maintained until day 22. Meannumber of counts from triplicate experiments is displayed with errorbars indicating S.D. b. Counts are shown for G418 resistant colonies (ina 32-cm² area) in Oct4/Sox2/Klf4/c-Myc over-expressing MEFs cultured inES cell media, Wnt3a over-expressing conditioned media, and Wnt3aover-expressing conditioned media with ICG-001 (4 μM). Selection wasinitiated on day 10 poet-induction, Wnt3a-CM was maintained until day17, and colonies were assessed on day 20. c. Wnt stimulation promotesthe formation of iPS cells in the absence of c-Myc transduction. Thiscould be due to: i) direct regulation by the Wnt pathway of keyendogenous pluripotency factors, such as Oct4, Sox2 and Nanog assuggested by genomic studies in ES cells (Cole et al., 2008), ii) Wntpathway-induced activation of endogenous Myc (He et al., 1998; Cole etal., 2008), or other cell proliferation genes, accelerating thesequential process of forming iPS colonies.

FIG. 4. (a) Timeline of initial experiments showing ability of Wnt3aconditioned medium to promote generation of iPS cells. Expression of thepluripotency-inducing factors was induced on day 2. Expression of GFPand colony formation were assessed as indicated (b). Wnt3a promotes iPScell formation in cells over-expressing Oct4, Sox2, Klf4 and c-Myc; FIG.4C Wnt3a promotes iPS cell formation in cells over-expressing Oct4,Sox2, Klf4 without engineered expression of c-Myc; (c) Wnt3a promotesiPS cell formation in cells over-expressing Oct4, Sox2, Klf4 withoutengineered expression of c-Myc.

FIG. 5. Structure of ICG-001.

DETAILED DESCRIPTION OF THE INVENTION Introduction and Definitions

The present invention relates to compositions and methods forreprogramming somatic cells, e.g., for reprogramming somatic cells topluripotency in vitro. The invention provides methods for reprogrammingsomatic cells to a less differentiated state. The resulting cells arereferred to herein as “reprogrammed somatic cells” (“RSC”) herein, or insome embodiments as induced pluripotent stem (iPS) cells if reprogrammedto a pluripotent state. The term “somatic cell” refers to any cell otherthen a gem cell, a cell present in or obtained from e pre-implantationembryo, or a cell resulting from proliferation of such a cell in vitro.In some embodiments the somatic cell is a “non-embryonic somatic cell”,by which is meant a somatic cell that is not present in or obtained(torn an embryo and does not result from proliferation of such a cell invitro, to some embodiments the somatic cell is an “adult somatic cell”,by which is meant a cell that is present in or obtained from an organismother than an embryo or a fetus or results from proliferation of such acell in vitro. Unless otherwise indicated the methods for reprogrammingcell to a less differentiated state are performed in vitro, i.e., theyare practiced using isolated somatic cells maintained in culture.

The invention encompasses the recognition that naturally occurringsignaling molecules that modulate the expression of endogenous ES celltranscription factors are promising candidates for soluble agents thatenhance reprogramming. The invention also encompasses the recognitionthat modulating foe biological pathways with which such naturallyoccurring signaling molecules internet is of use to enhance (e.g.,increase speed and/or efficiency of) reprogramming. The invention alsoencompasses the recognition that agents (whether naturally occurring orsynthetic, e.g., small molecules) that modulate the biological pathwayswith which such naturally occurring signaling molecules interact, arepromising candidates for soluble agents that enhance reprogramming.

As described in more detail below, certain embodiments of the inventionare based at least in pert on the recognition that modulating, e.g.,activating, the Wnt pathway is of use in reprogramming somatic cells.Certain of the methods comprising increasing activity of the Wnt pathwayin somatic cells such that at least some of the cell becomereprogrammed, e.g., to a pluripotent state. Certain of the methodscomprise culturing somatic cells in Wnt conditioned medium such that atleast some of the cells become reprogrammed, e.g., to a pluripotentstate.

Reprogramming, as used herein, refers to a process that alters orreverses the differentiation state of a somatic cell. The cell can beeither partially or terminally differentiated prior to reprogramming.Reprogramming encompasses complete reversion of the differentiationstate of a somatic cell to a pluripotent state. As known in the art, a“pluripotent” cell has the ability to differentiate into or give rise tocells derived from all three embryonic germ layers (endoderm, mesodermand ectoderm) and typically has the potential to divide in vitro for along period of time, e.g., greater than one year or more than 30passages. ES cells are an example of pluripotent cells. Reprogrammingalso encompasses partial reversion of the differentiation state of asomatic cell to a multipotent state. A “multipotent” cell is a cell thatis able to differentiate into some hot not all of the cells derived fromall three germ layers. Thus, a multipotent cell is a partiallydifferentiated cell. Adult stem cells are multipotent cells. Adult stemcells include, for example, hematopoietic stem cells and neural stemcells. Reprogramming also encompasses partial reversion of thedifferentiation state of a somatic cell to a state that renders the cellmore susceptible to complete reprogramming to a pluripotent state whensubjected to additional manipulations such as those described herein.Such contacting may result in expression of particular genes by thecells, which expression contributes so reprogramming. In certainembodiments of the invention, reprogramming of a somatic cell causes thesomatic cell to assume a pluripotent, ES-like state. The resulting cellsare referred to herein as reprogrammed pluripotent somatic cells orinduced pluripotent stem (iPS) cells.

Reprogramming involves alteration, e.g., reversal, of at least some ofthe heritable patterns of nucleic acid modification (e.g., methylation),chromatin condensation, epigenetic changes, genomic imprinting, etc.,that occur during cellular differentiation as a zygote develops into anadult Reprogramming is distinct from simply maintaining the existingundifferentiated state of a cell that is already pluripotent ormaintaining the existing less than fully differentiated state of a cellthat is already a multipotent cell (e.g., a hematopoietic stem cell).Reprogramming is also distinct (tom promoting the self-renewal orproliferation of cells that are already pluripotent or multipotent,although the compositions and methods of the invention may also be ofuse for such purposes. Certain of the compositions and methods of thepresent invention contribute to establishing the pluripotent state. Themethods may be practiced on cells that fully differentiated and/orrestricted to giving rise only to cells of that particular type, ratherthan on cells that are already multipotent or pluripotent.

Somatic cells are treated many of a variety of ways to causereprogramming according to the methods of the present invention. Thetreatment can comprise contacting the cells with one or more agent(s)that contribute to reprogramming (“reprogramming agent”). Suchcontacting may be performed by maintaining the cell in culture mediumcomprising the agent(s). In some embodiments the somatic cells aregenetically engineered. The somatic cell may be genetically engineeredto express one or more reprogramming agents as described further below.

In the methods of the present invention somatic cells may, in general,be cultured under standard conditions of temperature, pH, and otherenvironmental conditions, e.g., as adherent cells in tissue cultureplates at 37° C. in an atmosphere containing 5-10% CO₂. The cells and/orthe culture medium are appropriately modified to achieve reprogrammingas described herein, to certain embodiments, the somatic cells arecultured on or in the presence of a material that mimics one or morefeatures of the extracellular matrix or comprises one or moreextracellular matrix or basement membrane components. In someembodiments Matrigel™ is used. Other materials include proteins ormixtures thereof such as gelatin, collagen, fibronectin, etc. In certainembodiments of the invention the somatic cells are cultured in thepresence of a feeder layer of cells. Such cells may, for example, be ofmurine or human origin. They may be irradiated, chemically inactivatedby treatment with a chemical inactivator such as mitomycin c, orotherwise treated to inhibit their proliferation if desired. In otherembodiments the somatic cells are cultured without feeder cells.

Generating pluripotent or multipotent cells by somatic cellreprogramming using the methods of the present invention has a number ofadvantages. First, the methods of the present invention allow one togenerate autologous pluripotent cells, which are cells specific to andgenetically matched with an individual. The cells are derived fromsomatic cells obtained from the individual. In general, autologous cellsare less likely than non-autologous cells to be subject to immunologicalrejection. Second, the methods of the present invention allow theartisan to generate pluripotent cells without using embryos, oocytes,and/or nuclear transfer technology. Applicants' results demonstrate that(i) somatic cells can be reprogrammed to an ES-tike state without theneed to engineer the cells to express an oncogene such as c-Myc; and(ii) reprogramming of somatic cells can at least in part be effected bymeans other than engineering the cells to express reprogramming factors,i.e., by contacting the cells with a reprogramming agent other than anucleic acid or viral vector capable of being taken up and causing astable genetic modification to the cells. In particular, the inventionencompasses the recognition that extracellular signaling molecules,e.g., molecules that when present extracellularly bind to cell surfacereceptors and activate intracellular signal transduction cascades, areof use to reprogram somatic cells. The invention Anther encompasses therecognition that activation of such signaling pathways by means otherthan the application of extracellular signaling molecules is also of useto reprogram somatic cells. In addition, the methods of the presentinvention enhanced the formation of colonies of BS-like cells that weredetectable based on morphological criteria, without the need to employ aselectable marker. The present disclosure thus reflects severalfundamentally important advances in the area of in vitro somatic cellreprogramming technology. While certain aspects of the invention areexemplified herein using Wnt pathway signaling, the methods of theinvention encompass activation of other signaling pathways for purposesof reprogramming somatic cells.

Definitions of certain terms useful for understanding aspects of theinvention are presented below:

“Agent” as used herein means any compound or substance such as, but notlimited to, a small molecule, nucleic acid, polypeptide, peptide, drug,ion, etc.

A “cell culture medium” (also referred to herein as a “culture medium”or “medium”) is a medium for culturing edit containing nutrients thatmaintain cell viability and support proliferation. The cell culturemedium may contain any of the following in an appropriate combination:salt(s), buffer(s) amino acids, glucose or other sugar(s), antibiotics,serum or serum replacement, and other components such as peptide growthfactors, etc. Cell culture media ordinarily used for particular celltypes are known to those skilled in the art. Some son-limiting examplesare provided herein.

“Cell line” refers to a population of largely or substantially identicalcells that has typically been derived from a single ancestor cell orfrom a defined and/or substantially identical population of ancestorcells. The cell line may have been or may be capable of being maintainedin culture for an extended period (e.g., months, years, for an unlimitedperiod of time). It may have undergone a spontaneous or induced processof transformation conferring an unlimited culture lifespan on the cells.Cell lines include all those cell Knee recognized in the art as such. Itwill be appreciated that cells acquire mutations and possibly epigeneticcharges over time such that at least some properties of individual cellsof a cell line may differ with respect to each other.

The term “exogenous” refers to a substance present in a cell or organismother than its native source. For example, the terms “exogenous nucleicacid” or “exogenous protein” refer to a nucleic acid or protein that hasbeen introduced by a process involving the hand of man into s biologicalsystem such as a cell or organism in which it is not normally found orIn which it is found in lower amounts. A substance will be consideredexogenous if it is introduced into a cell or an ancestor of the cellthat inherits the substance. In contrast, the term “endogenous” refersto a substance feat is native to the biological system.

“Expression” refers to the cellular processes involved in producing RNAand proteins and as appropriate, secreting proteins, including whereapplicable, but not limited to, for example, transcription, translation,folding, modification and processing. “Expression products” include RNAtranscribed from a gene and polypeptides obtained by translation of mRNAtranscribed from a gene.

A “genetically modified” or “engineered” cell as used herein refers to acell into which an exogenous nucleic acid has been introduced by aprocess involving the hand of man (or a descendant of such a cell feathas inherited at least a portion of the nucleic acid). The nucleic acidmay for example contain a sequence that is exogenous to the cell, it maycontain native sequences (i.e., sequences naturally found in the cells)but in a non-naturally occurring arrangement (e.g., a coding regionlinked to a promoter from a different gene), or altered versions ofnative sequences, etc. The process of transferring the nucleic into thecell can be achieved by any suitable technique. Suitable techniquesinclude calcium phosphate or lipid-mediated transfection,electroporation, and transduction or infection using a viral vector. Insome embodiments the polynucleotide or a portion thereof is integratedinto the genome of the cell. The nucleic acid may have subsequently beenremoved or excised from the genome, provided that such removal orexcision results in a detectable alteration in the cell relative to anunmodified but otherwise equivalent cell.

“Identity” refers to the extent to which the sequence of two or morenucleic acis or polypeptides is the same. The percent identity between asequence of interest and a second sequence over a window of evaluation,e.g., over the length of the sequence of interest, may be computed byaligning the sequences, determining the number of residues (nucleotidesor amino acids) within the window of evaluation that are opposite anidentical residue allowing the introduction of gaps to maximizeidentity, dividing by the total number of residues of the sequence ofinterest or the second sequence (whichever is greater) dial fill withinthe window, and multiplying by 100. When computing the number ofidentical residues needed to achieve a particular percent identity,fractions are to be rounded to the nearest whole number. Percentidentity can be calculated with the use of a variety of computerprograms known in the art. For example, computer programs such asBLAST2, BLASTN, BLASTP, Gapped BLAST, etc., generate alignments andprovide percent identity between sequences of interest. The algorithm ofKarlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci. USA87:22264-2268, 1990) modified as in Karlin and Altschul, Proc. Natl.Acad. Sci. USA 90:5873-5877, 1993 is incorporated into the NBLAST andXBLAST programs of Altschul et al. (Altschul, et al., J. Mol. Biol.215:403-410, 1990). To obtain gapped alignments for comparison proposes,Gapped BLAST is utilized as described in Altschul et al. (Altschul, etal. Nucleic Acids Res. 25: 3389-3402, 1997). When utilizing BLAST andGapped BLAST programs; the default parameters of the respective programsmay be used. A PAM250 or BLOSUM62 matrix may be used. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (NCBI). See the Web site having URLwww.ncbi.nlm.nih.gov for these programs. In a specific embodiment,percent identity is calculated using BLAST2 with default parameters asprovided by the NCBI.

“Isolated” or “partially purified” as wed herein refers, in the case ofa nucleic acid or polypeptide, to a nucleic acid or polypeptideseparated from at least one other component (e.g., nucleic acid orpolypeptide) that it present with the nucleic acid or polypeptide asfound in its natural source and/or that would be present with thenucleic acid or polypeptide when expressed by a cell, or secreted in thecase of secreted polypeptides. A chemically synthesized nucleic acid orpolypeptide or one synthesized using in vitro transcription/translationis considered “isolated”. An “isolated cell” is a cell that has beenremoved from an organism in which it was originally found or adescendant of such a cell. Optionally the cell has been cultured hivitro, e.g., in the presence of other cells. Optionally the cell islater introduced into a second organism or reintroduced into theorganism from which it (or the cell from which it is descended) wasisolated.

The term “gene whose function is associated with pluripotency”, as usedherein, refers to a gene whose expression under normal conditions (e.g.,in the absence of genetic engineering or other manipulation designed toalter gene expression) occurs in and is typically restricted topluripotent stem cells, and is crucial for their functional identity assuch. It will be appreciated that the polypeptide encoded by a genefunctionally associated with pluripotency may be present as a maternalfactor in the oocyte. The gene may be expressed by at least some cellsof the embryo, e.g., throughout at least a portion of thepreimplantation period and/or in germ cell precursors of the adult.

“Modulate” is used consistently with its use in the art, i.e., meaningto cause or facilitate a qualitative or quantitative change, alteration,or modification in a process, pathway, or phenomenon of interest.Without limitation, such change may be an increase, decrease, or changein relative strength or activity of different components or branches ofthe process, pathway, or phenomenon. A “modulator” is an agent thatcauses or facilitates a qualitative or quantitative change, alteration,or modification in a process, pathway, or phenomenon of interest.

The term “pluripotency factor” is used refer to the expression productof a gene whose function is associated with pluripotency, e.g., apolypeptide encoded by the gene. In some embodiments the pluripotencyfactor is one that is normally substantially not expressed in somaticcell types that constitute the body of an adult animal (with theexception of germ cells or precursors thereof). For example, thepluripotency Actor may be one whose average level in ES edit is at least50-fold or 100-fold greater then its avenge level in those terminallydifferentiated cell types present in the body of an adult mammal. Insome embodiments, the pluripotency factor is one that is essential tomaintain the viability or pluripotent state of ES cells in vivo and/orES cells derived using conventional methods. Thus if the gene encodingthe factor is knocked out or inhibited (i.e., its expression iseliminated or substantially reduced), the ES cells are not formed, dieor, in some embodiments, differentiate. In some embodiments, inhibitingexpression of a gene whose function is associated with pluripotency inan ES cell (resulting in, e.g., a reduction in the average steady statelevel of RNA transcript and/or protein encoded by the gene by at least50%, 60%, 70%, 80%, 90%, 95%, or more) insults in a cell that is viablebut no longer pluripotent. In some embodiments the gene is characterizedin that its expression in an ES cell decreases (resulting in, e.g., areduction m the average steady state level of RNA transcript and/orprotein encoded by the gene by at least 50%, 60%, 70%, 80%, 90%, 95%, ormore) when the cell differentiates into a terminally differentiatedcell.

A “pluripotency inducing gene”, as used herein, refers to a gene whoseexpression, contributes to reprogramming somatic cells to a pluripotentstate. “Pluripotency inducing factor” refers to an expression product ofa pluripotency inducing gene. A pluripotency inducing factor may, butneed not be, a pluripotency factor. Expression of an exogenouslyintroduced pluripotency inducing factor may be transient, i.e., it maybe needed during at least a portion of the reprogramming process inorder to induce pluripotency and/or establish a stable pluripotent stelebut afterwards not required to maintain pluripotency. For example, thefactor may induce expression of endogenous genes whose function isassociated with pluripotency. These genes may then maintain thereprogrammed cells in a pluripotent state.

“Polynucleotide” is used herein interchangeably with “nucleic acid” toindicate a polymer of nucleosides. Typically a polynucleotide of thisinvention is composed of nucleosides that are naturally found in DNA orRNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine,deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine)joined by phosphodiester bonds. However the term encompasses moleculescomprising nucleosides or nucleoside analogs containing chemically orbiologically modified bases, modified backbones, etc., whether or notfound in naturally occurring nucleic acids, and such molecules may bepreferred for certain applications. Where this application refers to apolynucleotide it is understood that both DNA, RNA, and in each caseboth single- and double-stranded forms (and complements of eachsingle-stranded molecule) are provided. “Polynucleotide sequence” asused herein can refer to the polynucleotide material itself and/or tothe sequence information (i.e. the succession of letters used asabbreviations for bases) that biochemically characterizes a specificnucleic acid. A polynucleotide sequence presented herein is presented ina 5′ to 3′ direction unless otherwise indicated.

“Polypeptide” refers to a polymer of amino acids. The terms “protein”and “polypeptide” are used interchangeably heroin. A peptide is arelatively short polypeptide, typically between about 2 and 60 aminoacids in length. Polypeptides used herein typically contain amino acidssuch as the 20 L-amino acids that are most commonly found in proteins.However, other amino acids and/or amino acid analogs known in the artcan be used. One or more of the amino acids in a polypeptide may bemodified, for example, by the addition of a chemical entity such as acarbohydrate group, a phosphate group, a fatty acid group, a linker forconjugation, functionalization, etc. A polypeptide that has anonpolypeptide moiety covalently or noncovalently associated therewithis still considered a “polypeptide”. Exemplary modifications includeglycosylation and palmitoylation. Polypeptides may be purified fromnatural sources, produced using recombinant DMA technology, synthesizedthrough chemical means such as conventional solid phase peptidesynthesis, etc. The term “polypeptide sequence” or “amino acid sequence”as used herein can refer to the polypeptide material itself and/or tothe sequence information (i.e., the succession of letters or threeletter codes used as abbreviations for amino add names) thatbiochemically characterizes a polypeptide. A polypeptide sequencepresented herein is presented in an N-terminal to C-terminal directionunless otherwise indicated.

“Polypeptide variant” refers to any polypeptide differing from anaturally occurring polypeptide by amino acid insertion(s), deletion(s),and/or substitution(s), Variants may be naturally occurring or createdusing, e g, recombinant DNA techniques or chemical synthesis. In someembodiments amino acid “substitutions” are the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, i.e., conservative amino acid replacements.“Conservative” amino acid substitutions may be made on the basis ofsimilarity in any of a variety or properties such as side chain size,polarity, charge, solubility, hydrophobicity, hydrophilicity, and/oramphipathicity of the residues involved. For example, the non-polar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,glycine, proline, phenylalanine, tryptophan and methionine. The polar(hydrophilic), neutral amino acids include serine, threonine, cysteine,tyrosine, asparagine, and glutamine. The positively charged (basic)amino acids include arginine, lysine and histidine. The negativelycharged (acidic) amino acids include aspartic acid and glutamic acid.Insertions or deletions may range in size from about 1 to 20 aminoacids, e.g., 1 to 10 amino acids. In some instances larger domains maybe removed without substantially affecting function. In certainembodiments of the invention the sequence of a variant can be obtainedby malting no more than a total of 5, 10, 15, or 20 amino acidadditions, deletions, or substitutions to the sequence of a naturallyoccurring enzyme. In some embodiments not more than 1%, 5%, 10%, or 20%of the amino acids in a polypeptide are insertions, deletions, orsubstitutions relative to the original polypeptide. Guidance indetermining which amino acid residues may be replaced, added, or deletedwithout eliminating or substantially reducing activities of interest,may be obtained by comparing the sequence of the particular polypeptidewith that of homologous polypeptides (e.g., from other organisms) andminimizing the number of amino acid sequence changes made in regions ofhigh homology (conserved regions) or by replacing amino acids with thosefound in homologous sequences since amino acid residues that areconserved among various species are more likely to be important foractivity than amino acids that are not conserved.

“Purified” or “substantially purified” as used herein denote that theindicated nucleic acid or polypeptide is present in the substantialabsence of other biological macromolecules, e.g., polynucleotides,proteins, and the like. In one embodiment, the polynucleotide orpolypeptide is purified such that it constitutes at least 90% by weight,e.g., at least 9556 by weight, e.g., at least 99% by weight, of thepolynucleotide(s) or polypeptide(s) present (but water, buffers, ions,and other small molecules, especially molecules having a molecularweight of leas than 1000 daltons, can be present).

“RNA interference” is used herein consistently with its meaning in theart to refer to a phenomenon whereby double-stranded RNA (dsRNA)triggers the sequence-specific degradation or translational repressionof a corresponding mRNA having complementarity to a strand of the dsRNA.It will be appreciated that the complementarity between the strand ofthe dsRNA and the mRNA need not be 100% but need only be sufficient tomediate inhibition of gene expression (also referred toss “silencing”or“knockdown”). For example, the degree of complementarity is such thatthe strand can either (i) guide cleavage of the mRNA in the RNA-inducedsilencing complex (RISC); or (ii) cause translational repression of themRNA. In certain embodiments the double-stranded portion of the RNA isless than about 30 nucleotides in length, e.g., between 17 and 29nucleotides in length. In mammalian cells, RNAi may be achieved byintroducing an appropriate double-stranded nucleic acid into the cellsor expressing a nucleic acid in cells that is then processedintracellularly to yield dsRNA therein. Nucleic acids capable ofmediating RNAi are referred to herein as “RNAi agents”. Exemplarynucleic acids capable of mediating RNAi area short hairpin RNA (shRNA),a short interfering RNA (siRNA), and a microRNA precursor. These termsare well known and are used herein consistently with their meaning inthe art. siRNAs typically comprise two separate nucleic acid strandsthat are hybridized to each other to form a duplex. They can besynthesized. In vitro, e.g., using standard nucleic acid synthesistechniques. They can comprise a wide variety of modified nucleosides,nucleoside analogs and can comprise chemically or biologically modifiedbases, modified backbones, etc. Any modification recognized in the artas being useful for RNAi can be used. Some modifications result inincreased stability, cell uptake, potency, etc. In certain embodimentsthe siRNA comprises a duplex about 19 nucleotides in length and one ortwo 3′ overhangs of 1-5 nucleotides in length, which may be composed ofdeoxyribonucleotides. shRNA comprise single nucleic acid strand thatcontains two complementary portions separated by a predominantlynon-selfcomplementary region. The complementary portions hybridize toform a duplex structure and the non-selfcomplementary region forms aloop connecting the 3′ end of one strand of the duplex and the 5′ end ofthe other strand. shRNAs undergo intracellular processing to generatesiRNAs.

MicroRNAs (miRNAs) are small, non-coding, single-stranded RNAs of about21-25 nucleotides (in mammalian systems) that inhibit gene expression ina sequence-specific manner. They are generated intracellularly fromprecursors having a characteristic secondary structure comprised of ashort hairpin (about 70 nucleotides in length) containing a duplex thatoften includes one or more regions of imperfect complementarity.Naturally occurring miRNAs are only partially complementary to theirtarget mRNA and typically act via transitional repression. RNAi agentsmodelled on endogenous microRNA precursors are of use in the invention.In some embodiments, a sequence encoding the stem portion of a stem-loopstructure or encoding a complete stem-loop can be inserted into anucleic acid comprising at least a portico of an endogenous microRNAprimary transcript, e.g., in place of the sequence that encodes theendogenous microRNA or minimum (˜70 nucleotide) microRNA hairpin.

“Reprogramming factor” refers to a gene, RNA, or protein that promotesor contributes to cell reprogramming, e.g., in vitro. In aspects of theinvention relating to reprogramming factor(s), the invention providesembodiments in which the reprogramming factor(s) are of interest forreprogramming somatic cells to pluripotency in vitro. Examples ofreprogramming factors of interest for reprogramming somatic cells topluripotency in vitro are Oct4, Nanog, Sox2, Lin28, Klf4, c-Myc, and anygene/protein that can substitute for one or more of these in a method ofreprogramming somatic cells in vitro. “Reprogramming to a pluripotentstate in vitro”, or “reprogramming to pluripotency in vitro”, is usedherein to refer to in vitro reprogramming methods that do not requireand typically do not include nuclear or cytoplasmic transfer or cellfusion, e.g., with oocytes, embryos, gam cells, or pluripotent cells.Any embodiment or claim of the invention may specifically excludecompositions or methods relating to or involving nuclear or cytoplasmictransfer or cell fusion, e.g., with oocytes, embryos, germ cells, orpluripotent cells.

“Selectable marker” refers to a gene, RNA, or protein that whenexpressed, confers upon cells s selectable phenotype, such as resistanceto a cytotoxic or cytostatic agent (e.g., antibiotic resistance),nutritional prototrophy, or expression of a particular protein that canbe used as a basis to distinguish cells that express the protein fromcells that do not. Proteins whose expression can be readily detectedsuch as a fluorescent or luminescent protein or an enzyme that acts on asubstrate to produce a colored, fluorescent, or luminescent substance(“detectable markers”) constitute a subset of selectable markets. Thepresence of a selectable marker linked to expression control elementsnative to a gene that is normally expressed selectively or exclusivelyin pluripotent cells makes it possible to identify and select somaticcells that have been reprogrammed to a pluripotent state. A variety ofselectable marker genes can be used, such as neomycin resistance gene(neo), puromycin resistance gene (pure), guanine phosphoribosyltransferase (gpt), dihydrofolate reductase (DHPR), adenosine deaminase(ada), puromycin-N-acetyltransferase (PAC), hygromycin resistance gene(hyg), multidrug resistance gene (mdr), thymidine kinase (TK),hypoxanthine-guanine phosphoribosyltransferase (HPRT), and hisD gene.Detectable markers include green fluorescent protein (GFP) blue,sapphire, yellow, red, orange, and cyan fluorescent proteins andvariants of any of these. Luminescent proteins such as luciferase (e.g.,firefly or Renilla luciferase) are also of use. As will be evident toone of skill in the art, the term “selectable marker” as used herein canrefer to a gene or to an expression product of the gene, e.g., anencoded protein.

In some embodiments the selectable marker confers a proliferation and/orsurvival advantage on cells that express it relative to cells that donot express it or that express it at significantly lower levels. Suchproliferation and/or survival advantage typically occurs when the cellsare maintained under certain conditions, i.e., “selective conditions”.To ensure an effective selection, a population of cells can bemaintained for a under conditions and for a sufficient period of timesuch that cells that do not express the marker do not proliferate and/ordo not survive and are eliminated from the population or their number isreduced to only a very small friction of the population. The process ofselecting cells that express a marker that confers a proliferationand/or survival advantage by maintaining a population of cells underselective conditions so as to largely or completely eliminate cells thatdo not express the marker is referred to hereto as “positive selection”,and the marker is said to be “useful for positive selection”. Negativeselection and marker useful for negative selection are also of interestm certain of the methods described herein. Expression of such markersconfers a proliferation and/or survival disadvantage on cells thatexpress the marker relative to cell that do not express the marker orexpress it at significantly lower levels (or, considered another way,cells that do not express the marker have a proliferation and/orsurvival advantage relative to cells that express the marker). Cellsthat express the marker can therefore be largely or completelyeliminated from a population of cells when maintained in selectiveconditions for a sufficient period of time.

The terms “treat”, “treating”, “treatment”, etc., as applied to anisolated cell, include subjecting the cell to any kind of process orcondition or performing any kind of manipulation or procedure on thecell. As applied to a subject, the terms refer to providing medical orsurgical attention, care, or management to an individual. The individualis usually ill or injured, or at increased risk of becoming ill relativeto an average member of the population and in need of such attention,care, or management.

The term “Wnt”, or “Wnt protein” as used herein refers to a polypeptidehaving a naturally occurring amino acid sequence of a Wnt protein or afragment, variant, or derivative thereof that at least in part retainsthe ability of the naturally occurring protein to bind to Wnt receptors)and activate Wnt signaling. In addition to naturally-occurring allelicvariants of the Wnt sequences that may exist in the population, it willbe appreciated that, as is the case for virtually all proteins, avariety of changes can be introduced into the sequences listed under theaccession numbers in Table 1 (referred to as “wild type” sequences)without substantially altering the functional (biological) activity ofthe polypeptides. Such variants are included within the scope of theterm “Wnt”, “Wnt protein”, etc.

The variant could be, e.g., a polypeptide at least 80%, 85%, 90%, 95%,98%, or 99% identical to frill length Wnt. The variant could be afragment of fully length Wnt. The variant could be a naturally occurringsplice variant. The variant could be a polypeptide at least 80%, 83%,90%, 95%, 98%, or 99% identical to a fragment of Writ, wherein thefragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% aslong as the full length wild type polypeptide or a domain thereof havingan activity of interest such as the ability to bind to a Wnt receptor.In some embodiments the domain is at least 100, 200, 300, or 400 aminoacids in length, beginning at any amino acid position in the sequenceand extending toward the C-terminus. Variations known in the art toeliminate or substantially reduce the activity of the Wnt protein arepreferably avoided. In some embodiments, the variant lacks an N- and/orC-terminal portion of the fell length polypeptide, e.g., up to 10, 20,or 50 amino acids from either terminus is lacking. In some embodimentsthe polypeptide has the sequence of a mature Wnt polypeptide, by whichis meant a Wnt polypeptide that has had one or more portions such as asignal peptide removed during normal intracellular proteolyticprocessing (e.g., during co-translational or post-translationalprocessing). In some embodiments wherein the Wnt protein is producedother than by purifying it from cells that naturally express it, theprotein is a chimeric polypeptide, by which is meant that it containsportions from two or more different species, lit some embodimentswherein the Wnt protein is produced other than by purifying it fromcells that naturally express it, the protein is a Wnt derivative, by Wntis meant that the protein comprises additional sequences not related toWnt so long as those sequences do not substantially reduce thebiological activity of the protein.

One of skill in the art will be aware of, or will readily be able toascertain, whether a particular Wnt variant, fragment, or derivative isfunctional using assays known in the art. For example, the ability of avariant of a Wnt polypeptide to bind to a Wnt receptor can be assessedusing standard protein binding assays. Convenient assays includemeasuring the ability to activate transcription of a reporter constructcontaining a TCP binding site operably linked to a nucleic acid sequenceencoding a delectable marker such as luciferase. One assay involvesdetermining whether the Wnt variant induces phosphorylation ofβ-catenin. Phosphorylation status can be determined using any suitablemethod, e.g., immunoblotting. Other assays involve testing fee variantor fragment for known biological activities of Wnt. See, e.g., Barker,N. and Clevers, H., Nat Rev Drag Discov. 5(12):997-1014, 2006, whichdescribes assays suitable for identifying agents that modulate Wntpathway activity. Such essay may readily be adapted to identity orconfirm activity of agents that activate Wnt pathway activity. Incertain embodiments of the invention a functioned variant or fragmenthas at least 50%, 60%, 70%, 80%, 90%, 95% or more of the activity of thefull length wild type polypeptide.

“Wnt pathway activity” or “Wnt signaling” refers to the series ofbiochemical events that ensues following binding of a stimulatory ligand(e.g., a Wnt protein) to a receptor for a Wnt family member, ultimatelyleading to changes in gene transcription and, if in vivo, often leadingto a characteristic biological effect in an organism.

Reprogramming Somatic Cells by Activating the Wnt Pathway

The present invention provides the recognition that activating the Wntpathway is of use to reprogram somatic cells. The invention providestire additional recognition that activating the Wnt pathway increasesthe efficiency of reprogramming of somatic cells, e.g., when such cellsare subjected to a treatment that would result in reprogramming of atleast some cells. “Increase the efficiency of reprogramming” means tocause an increase in the percentage of cells that undergo reprogrammingwhen a population of cells is subjected to a reprogramming treatment,typically resulting in a greater number of individual colonies ofreprogrammed cells after a given time period. In some embodiments of theinvention, activating the Wnt pathway according to the inventionincreases the number of reprogrammed cells and/or the number of coloniesof reprogrammed cells and/or the percentage of cells that undergoreprogramming. The invention further provides the recognition thatactivating the Wnt pathway enables reprogramming of somatic cells thathave not been genetically modified to increase their expression of anoncogene such as c-Myc. The invention thus provides ways to substitutefor engineered expression of c-Myc in any method of reprogrammingsomatic cells that would otherwise involve engineering cells to expressc-Myc. In some embodiments of the invention, activating the Wnt pathwayis sufficient to allow reprogramming under conditions in whichreprogramming would not otherwise occur.

The invention provides methods for generating reprogrammed somatic editcomprising modulating, e.g., increasing, activity of the Wnt pathway.The invention further provides compositions of use in the methods. Inone aspect, the invention provides a method of reprogramming a somaticcell comprising modulating, e.g., increasing Wnt pathway activity in thecell. The invention farther provides improved methods for reprogrammingof somatic cells, the method comprising subjecting somatic cells to atreatment that may reprogram at least some of the cells, wherein theimprovement comprises increasing the activity of a Wnt pathway in saidcells. The treatment may be any treatment known in the art to be of useto reprogram somatic cells or considered to be of potential use far thispurpose. In certain embodiments of the invention Wet pathway activity isincreased using activators of the Wnt pathway such as small molecules,soluble Wnt proteins, or agents that mediate RNA interference andthereby inhibit endogenous inhibitors of the Wnt pathway. In certainembodiments somatic cells to be reprogrammed are cultured in Wntconditioned medium. In any of the embodiments of the invention, unlessotherwise indicated or evident from the context, “reprogramming” canrefer to reprogramming to a pluripotent state.

Wnts are a family of secreted proteins important for a wide array ofdevelopmental and physiological processes (Mikels, A J and Nusse, R.,Oncogene, 25: 7461-7468, 2006). Wnts are related to one another insequence and strongly conserved in structure and function acrossmultiple species. Thus a Wnt protein displaying activity in one speciesmay be used in other species to activate the Wnt pathway in such speciesand may be expected to display similar activity. Wnt family membersinclude Wnt1, Wnt2, Wnt2b (also called Wnt13), Wnt3, Wnt3a, Wnt4, Wnt5a,Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt5c, Wnt10a,Wnt10b, Wnt11, Wnt14, Wnt15, or Wnt16. Sequences of Wnt genes andproteins are known in the art. One of skill in the art can readily findthe Gene ID, accession numbers, and sequence information for Wnt familymembers and other genes and proteins of interest herein in publiclyavailable databases (see Table 1 for examples).

TABLE 1 Wnt pathway proteins, effectors, and regulators Gene Gene IDAccession numbers (mRNA/protein) Wnt3a (mouse) 22416 NM_009522/NP_033548Wnt3a (human) 89780 NM_033131/NP_149122 β-catenin (mouse) 12387NM_007614/NP_031640 β-catenin (human) 1499 NM_001098209/NP_001091679NM_001098210/NP_001091680 NM_001904/NP_001895 GSK3α (mouse) 606496NM_001031667/NP_001026837 GSK3α (human) 2931 NM_019884/NP_063937 GSK3β(mouse) 56637 NM_019827/NP_062801 GSK3β (human) 605004NM_002093/NP_002084 Sox2 (mouse) 20674 NM_011443/NP_035573 Sox2 (human)6657 NM_003106/NP_003097 KIf4 (mouse) 16600 NM_010637/NP_034767 KIf4(human) 9314 NM_004235/NP_004226 Oct4 (mouse) 18999 NM_013633/NP_038661Oct4 (human) 5460 NM_203289/NP_976034 Oct4 (human) 5460NM_002701/NP_002692 Nanog (mouse) 71950 NM_028016.2/NP_082292.1 Nanog(human) 79923 NM_024865/NP_079141 Lin28 (mouse) 83557NM_145833/NP_665832 Lin28 (human) 79727 NM_024674/NP_078950

Wnt signaling is initiated by interaction of Wnt proteins with a varietyof receptors, including members of the Frizzled (Fz) family oftransmembrane receptors and members of the low-density-lipoproteinreceptor-related protein (LRP) family (e.g., LRP5/LRP6). Theextracellular Wnt signal stimulates intracellular signal transductioncascades including the canonical pathway, which regulates geneexpression in the nucleus (reviewed by Logan C Y and Nusse, R. Annu,Rev. Cell Dev. Biol., 20:781-810, 2004) and several non-canonicalpathways (reviewed by Kohn, A D and Moon, R T, Cell Calcium, 38:439-446,2005). Briefly, Wnt signaling via the canonical pathway leads tostabilization and nuclear localization of β-catenin, which assembleswith members of the T-cell factor/lymphoid enhancer factor (TCF/LEF)family of transcription Actors to form complexes that generally activatetranscription. In the absence of Wnt signaling β-catenin is insteadtargeted for degradation by the β-catenin destruction complex, andTCF/LEFs form complexes that generally repress transcription. In theabsence of Wnt signaling, kinases such as glycogen synthase kinase-3(GSK3) and casein kinase 1 (CK1) phosphorylate β-catenin, which as aconsequence is ubiquinated and targeted for destruction by theproteasome. Activation of the Wnt pathway thus results in diminishedphosphorylation of β-catenin, thereby leading to its stabilization.Several endogenous proteins have been identified as inhibitors of Wntsignaling, including Dickkopf (Dkk), breakpoint cluster region protein(Bcr), proteins comprising a WIF (Wnt inhibitory Actor) domain etc.

In certain embodiments of the invention the reprogramming methodscomprise contacting a cell with an agent (that modulates, e.g.,increases, the activity of a Wnt pathway. In some embodiments,increasing the Wnt pathway induces the cell to become pluripotent andpossess features characteristic of ES cells. The methods are thus of useto generate pluripotent, ES-like cells (iPS cells). In certainembodiments of the invention a treatment that causes increased activityof a Wnt pathway is one that results in increased intracellular levelsof β-catenin. In certain embodiments of the invention, a treatment thatcauses increased activity of a Wnt pathway is one that results inincreased nuclear translocation of β-catenin. In certain embodiments ofthe invention, a treatment that causes increased activity of a Wntpathway is one capable of causing changes. In gene expressioncharacteristic of cells exposed to a source of biologically active Wntprotein. In some embodiments of the invention, reprogramming ismodulated using e Wnt pathway inhibitor.

A considerable advance towards the goal of reprogramming somatic cellsto a pluripotent state in vitro was achieved when it was shown that celllines with some of the properties of ES cells could be produced byintroducing genes encoding four transcription factors associated withpluripotency, i.e., Oct3/4, Sox2, c-Myc and Klf4, into mouse skinfibroblasts via retroviral infection, and then selecting cells thatexpressed a marker of pluripotency, Fbx15, in response to these factors(Takahashi, K. & Yamanaka, S. Cell 126, 663-676, 2006). However, theresulting cells differed from ES cells in their gene expression and DNAmethylation patterns and when injected into normal mouse blastocysts didnot result in live chimeras (animals carrying cells throughout theirbodies from both the original blastocyst and from the introduced cells).Subsequent work improved on these results by performing more rigorousselect ion, resulting in derivation of stable reprogrammed cell linesthat, based on reported transcriptional, imprinting (expression ofelides predetermined by die parent from which they originated) andchromatin-modification profiles, appeared essentially identical to EScells (Okita, K., et al., 448, 313-317, 2007; Wernig, M. et al. Nature448, 318-324, 2007; Maherali, N. et al. Cell Stem Cell 1, 33-70, 2007).Somatic cells that have been reprogrammed to a pluripotent state invitro using these methods or other methods (e.g., involving applicationof small molecules) are referred to herein consistently with usage inthe art as “induced plan potent stem” (iPS) cells. Subsequently, it wasshown that human somatic cells can also be reprogrammed to pluripotencyusing these factors. Furthermore, it was demonstrated that thecombination of Oct4, Nanog, Sox2, and Lin28 was also able to reprogramsomatic cells to a pluripotent state in vitro (Yu J, Science, 318(5858):1917-20, 2007). However, generation of these cells also Involvedengineering the cells to express multiple transcription factors andemployed retroviral transduction.

Applicants have now shown that an increased number of colonies comprisedof ES-like cells developed when somatic cells genetically engineered toexpress Oct4, Sox2, Klf4, and c-Myc were cultured with Wnt3a conditionedmedium than when the cells were cultured in medium conditioned bycontrol cells or in standard cell culture medium conventionally used forthe propagation of ES cells. Applicants further showed that coloniescomprised of ES-like cells developed when somatic cells engineered toexpress Oct4, Sox2, and Klf4 but not modified to express c-Myc werecultured in Wnt3a conditioned medium, whereas colonies of ES-like cellsdid not form within the 20 day time period shown in FIG. 1 when suchcells were cultured in unconditioned medium or medium conditioned bycontrol cells. In both cases, the colonies displayed morphologicalfeatures characteristic of ES cell colonies and expression of adetectable marker indicative of Oct4 expression. By all criteria tested,the cells appear to be pluripotent, ES-like cells (iPS cells).Furthermore, culturing the somatic cells in Wnt3a conditioned mediumappeared to select for reprogrammed cells. The colonies formed in thepresence of Wnt3a conditioned medium appeared more homogenous than thoseobtained in the absence of Wnt3a conditioned medium. The methods arethus of use to facilitate identification of reprogrammed cells, andoptionally to facilitate separation of such cells from cells that havenot become reprogrammed, without the need for chemical selection relyingon so introduced genetic element such as a gene whose expression productconfers drug resistance or fluorescence. The methods are thus of use togenerate reprogrammed cells that do not carry genetic modifications forpurposes of selection or detection of the reprogrammed cells.Furthermore, the methods are of use to increase the average percentageof reprogrammed cells in a colony comprising reprogrammed cells relativeto the average percentage of cells that would be reprogrammed in theabsence of an agent that increases Wnt pathway activity.

Applicants and others have noticed that some iPS-like cells can formwithout infecting the cells with c-Myc virus. However, this is alow-efficiency event and could be at least in part a result ofinsertional mutagenesis wherein a viral integration event directlyactivates c-Myc or c-Myc target gene(s). In Applicants' experiments, atvery toe time points, some colonies were seen on the plates dun wereoverexpressing Klf4, Sox2 and Oct4 (without introducing c-Myc virus),even without Wnt conditioned medium. Wnt-conditioned mediumsignificantly reduced the time required and increased the efficiency ofthe reprogramming process. One aspect of the invention is that thefaster timing of reprogramming achieved using the methods of theinvention will facilitate the use of transient means of overexpressionof pluripotency Inducing factors for iPS formation (for example,transient transfection) and/or reprogramming by treating somatic cellswith reprogramming agents such as proteins, small molecules, etc.,instead of viral infection. In addition, Applicants propose thatincreased efficiency of iPS formation using the methods of the inventioncould be of particular use in reprogramming human cells, either with orwithout Myc overexpression.

Without limitation, the methods are thus of use to Increase the speed ofreprogramming somatic cells to iPS cells. Thus, the invention provides amethod of increasing the speed of reprogramming somatic cells,comprising culturing a population of somatic mammalian cells in Wntconditioned cell culture medium so that at least acme of the cells areinduced to become ES-like cells within a shorter period of time thanwould be the case in the absence of Wnt conditioned medium. Theinvention also provides a method of increasing the speed ofreprogramming somatic cells comprising activating the Wnt pathway in acultured population of somatic cells so that at least some of the cellsare induced to become ES-like cells within a shorter period of time thanwould be the case if the Wnt pathway was not activated. The inventionalso provides a method of increasing the speed of reprogramming somaticcells comprising culturing a population of somatic mammalian cells inthe presence of an agent that increases Wnt pathway activity so that atleast some of the cells are induced to become ES-like cells within ashorter period of time than would be the case in the absence of saidagent. In some embodiments of the invention, the period of time is 7days, while in other embodiments the period of time is 10, 15, or 20days. In some embodiments of the invention, the cells are treated (e.g.,genetically engineered) so that they express Sox2, Klf4, Oct4, and c-Mycat levels greater than would be the case in the absence of suchtreatment. In some embodiments of the invention, the cells are treatedso that they overexpress Sox2, Klf4, and Oct4 at levels greater thanwould be the case in the absence of such treatment, but are notgenetically engineered to overexpress c-Myc. One method of treatment isinfecting the cells with viruses (e.g., retrovirus, lentivirus) ortransfecting the cells with viral vectors (e.g., retroviral, lentiviral)that contain the sequences of the factors operably linked to suitableexpression control elements to drive expression in the cells followinginfection or transfection and, optionally integration into the genome asknown in the art. Further details regarding the compositions and methodsof the invention are provided below.

The invention provides a method of reprogramming a somatic cell,comprising culturing the cell in Wnt conditioned cell culture medium sothat the cell becomes reprogrammed. In some embodiments, culturing thecell in Wnt conditioned cell culture medium induces the cell to becomepluripotent and possess features characteristic of ES cells. The methodsare thus of use to generate pluripotent, ES-like cells (iPS cells). Insome embodiments, the Wnt conditioned cell culture medium comprisesWnt3a conditioned medium.

The term “conditioned medium” refers to cell culture medium that haspreviously been used for culturing cells. A conditioned medium ischaracterized in that it contains soluble substances, e.g., signalingmolecules, growth factors, hormones etc., which are produced by cellsduring their cultivation and released into the medium. As used herein,“Wnt conditioned medium” refers to conditioned medium that has beenpreviously used for culturing cells that produce and secrete Wnt. Themedium may be father described by reference to a particular Wnt proteinproduced by the cells. For example, “Wnt3a conditioned medium” refers toconditioned medium that has been previously used for culturing cellsthat produce Wnt3a. The cells may also produce other Writs in additionto the particular Wnt specifically referred to. Any embodiment of theinvention employing Wnt conditioned medium may employ Wnt3a conditionedmedium unless otherwise indicated.

It will be appreciated that certain Wnts have similar biologicalactivities to Wnt3a and/or are closely related in sequence to Wnt3a.Conditioned media prepared using cells that produce such Wnts are usedin certain embodiments of the invention.

Conditioned medium may be prepared by methods known in the art. Suchmethods typically comprise culturing a first population of cells in acell culture medium, and then harvesting the medium (typically withoutharvesting the cells). The harvested medium may be filtered to removecell debris, etc. The conditioned medium (containing components secretedinto the medium by the cells) may then be used to support the growth ofa second population of cells. The cells are cultured in the medium forsufficient time to allow adequate concentration of released factors suchas Wnt (and/or consumption of media components) to produce a medium thatsupports the reprogramming of somatic cells. In some embodiments, mediumis conditioned by culturing for 24 h at 37° C. However, longer orshorter periods can be used such as between 24 and 72 hours. The cellscan be used to condition multiple batches medium over additional cultureperiods, for as tong as the cells retain their ability to condition themedium in an adequate fashion for the desired purpose.

The medium in which the cells are cultured to produce conditioned mediummay be conventional cell culture medium capable of maintaining viabilityof the cells. In some embodiments, the medium ii chemically defined. Insome embodiments, the medium is similar or identical in composition tomedium conventionally used to culture embryonic stem cells of the samespecies as the somatic cells to be reprogrammed using the conditionedmedium. The base medium used for conditioning can have any of a numberof different compositions, depending in part on the types of cells used.The medium must be able to support culture of the cell line used for theconditioning of the medium. In some embodiments, medium also supportsculture of somatic cells prior to their being reprogrammed and,optionally, somatic cells that have been reprogrammed. However, theconditioned medium can be supplemented with other components, combinedwith other medium, etc., after conditioning so as to tender it suitablefor culturing somatic cells and reprogrammed somatic cells.

Suitable base media can be made from the following components:Dulbecco's modified Eagle's medium (DMEM), Invitrogen Cat. No.11965-092; Knockout Dulbecco's modified Eagle's medium (KO DMEM),Invitrogen Cat. No. 10829-018; Ham's F12/50% DMEM basal medium; 200 mML-glutamate, Invitrogen Cat No. 15039-027; non-essential amino acidsolution, Invitrogen Cat. No. 11140-050; beta-mercaptoethanol; humanrecombinant basic fibroblast growth factor (bFGF). Exemplaryserum-containing ES medium is made with 80% DMEM (typically KO DMEM),20% defined fetal bovine serum (FBS) not heat inactivated, 1%non-essential amino acids, 1 mM L glutamine, and 0.1 mMβ-mercaptoethanol. The medium is filtered and stored at 4° C. for nolonger than 2 weeks. Scrum-free ES medium may be prepared with 80% KODMEM, 20% scrum replacement, 1% non-essential amino acids, 1 mML-glutamine, and 0.1 mM β-mercaptoethanol and a serum replacement suchas Invitrogen Cat No. 10828-028. The medium is filtered and stored at 4°C. Before combining with the cells used for conditioning, human bFGF canbe added to a final concentration of 4 ng/mL. Stem Pro® hESC SFM(Invitrogen Cat. No. A1000701), a fully defined, serum- and feeder-freemedium (SFM) specialty formulated for the growth and expansion of humanembryonic stem cells, is of use.

The cells used to prepare the conditioned medium may naturally produceWnt. In some embodiments the cells used to prepare the medium aregenetically engineered to increase their expression of Wnt, e.g., bytransfecting them with a cDNA encoding Wnt, wherein the Wnt codingsequence is operably linked to expression control sequences active inthe cells. See, e.g., Cai, L, et al., Cell Res. 17:62-72, 2007. In someembodiments, the cells produce and secrete Wnt into their mediumresulting in medium having a concentration of between 100 ng/ml and 1000ng/ml Wnt protein. In some embodiments, the cells produce and secreteWnt into their medium resulting in medium having a concentration ofbetween 200 ng/ml and 500 ng/ml Wnt protein. Cells that overexpress Wntcould also be used as feeder cells for purposes of reprogramming somaticcells.

Conditioned medium may be combined with unconditioned medium prior touse. For brevity, the resulting medium is still referral to asconditioned medium if it comprises at least 5% conditioned medium byvolume. In some embodiments the amount (by volume) of conditioned mediumis at least 1096, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or more conditioned medium. In some embodiments, the amount ofconditioned medium is between about 50% and 75% by volume. Theunconditioned medium may be standard cell culture medium. In someembodiments the unconditioned medium is medium conventionally used forpropagating ES cells of the same species as the somatic cells to bereprogrammed.

The conditioned medium may be used immediately after being harvestedfrom the cells used to produce it or may be stored (e.g., at about 4° C.or frozen) prior to use. The median may be stored under conditions andfor a time period consistent with maintaining the ability of theconditioned medium to support reprogramming in the methods of theinvention. Without limitation, such conditions and time may beconsistent with maintaining at least 20% of the original biologicalactivity of secreted Wnt present in the medium, which may be assessedusing methods mentioned above. The conditioned medium may beconcentrated or otherwise processed, e.g., using standard methods,provided such concentration or processing is consistent with maintainingthe ability of the concentrate to support reprogramming when added tounconditioned medium. Without limitation, such concentration orprocessing may be consistent with maintaining at least 20% of theoriginal biological activity of secreted Wnt present in the medium. Asnoted in the Examples, Applicants' results suggest dial normalfibroblasts (not engineered to overexpress Wnt) may secrete factors,perhaps including Wnt3a, that promote reprogramming, raising thepossibility that somatic cells undergoing reprogramming in vitro, e.g.,cells in culture that have been treated with retrovirus or otherwiseengineered to express Oct4, Sox2, Klf4, and optionally c-Myc, maysecrete such factors and thus contribute to their own reprogramming. Incertain embodiments of the present invention, Wnt-conditioned medium hasa greater concentration of Wnt protein and/or Wnt pathway activatingactivity than would be the case when unmodified somatic cells, e.g.,fibroblasts, undergoing reprogramming are cultured in medium known inthe art to be useful for culturing somatic cells undergoingreprogramming. In some embodiments, such concentration and/or Wntpathway activating ability may be at least 1.5, 2, 5, 10, 20, or moretimes as great as present in medium in which control fibroblasts arecultured as described in Example 5.

Certain methods of the invention involve contacting a somatic cell invitro with one or mote defined agent(s) that modulate, e.g., increase,Wnt pathway activity. The cells may be maintained in standard cellculture medium known in the art. The agent(s) may be added to the mediumprior to using it to culture the cells or during cell culture. The term“defined agent” in this context means that the structure, sapience, oridentity of the agent that modulates, e.g., increases, Wnt pathwayactivity is known and/or the agent is chemically synthesized and/or theagent is (prior to addition to the medium) isolated or at leastpartially purified. For example, the agent may not be an uncharacterizedor unidentified component of conditioned medium, cell or tissue lysateor extract, cell cytoplasm or nuclear material, etc.

A variety of agents may be used to increase Wnt pathway activity. Suchagents are referred to herein as “Wnt pathway activators” or “Wntagonists”. The Wnt pathway activator may act directly by interactingwith a Wnt receptor or indirectly by interacting with one or moreintracellular components of the Wnt signaling pathway such as β-catenin,a kinase or phosphatase that acts on β-catenin, a transcription (actorthat assembles with β-catenin, etc. The activator may increaseexpression of Wnt or a Wnt pathway component such as β-catenin. Incertain embodiments the Wnt pathway activator increases activity of theWnt pathway to levels sufficient to enhance reprogramming of somaticcells. In certain embodiments of the invention the Wnt pathway activatorinhibits degradation of β-catenin, thereby enhancing reprogramming ofsomatic cells. In certain embodiments of the invention, it is ofinterest to inhibit the Wnt pathway in somatic cells or in reprogrammedsomatic cells. For example, Wnt pathway inhibitors can be used tocharacterize or explore the mechanism by which reprogramming occursand/or to identify reprogramming agents (e.g., agents that do not actvia the Wnt pathway). Furthermore, m certain embodiments of theinvention, Wnt pathway inhibitors (e.g., small molecules, siRNA,proteins, etc.) may be of use to facilitate differentiation ofreprogrammed, pluripotent cells to a desired cell type, e.g., in invitro differentiation protocols.

In certain embodiments of the invention, the Wnt pathway activator orinhibitor is s protein or smell molecule that binds to a Wnt receptor.For example, the Wnt pathway activator can be a soluble, biologicallyactive Wnt protein.

In some embodiments the concentration of Wnt protein added to the mediumis between 10 and 10,000 ng/ml, e.g., between 100 and 5,000 ng/ml, e.g.,between 1,000 and 2,500 ng/ml or between 2,500 and 5,000 ng/ml, orbetween 5,000 and 10,000 ng/ml.

As noted above certain Wnts have similar biological activities to Wnt3aand/or are closely related in sequence to Wnt3a. Such Wnts and/or agentsthat mimic the activity of such Wnts are used in certain embodiments ofthe invention.

The Wnt protein may be isolated from naturally occurring sources (e.g.,mammalian cells that naturally produce the protein), produced ineukaryotic or prokaryotic cells using recombinant expression technology,or chemically synthesized. Soluble, biologically active Wnt proteins maybe prepared in purified form using methods known in the art. See, e.g.,U.S. Pat. Pub. No. 20040248803 and Willert, K, et al., Nature,423:448-52, 2003. In certain embodiments the soluble, biologicallyactive Wnt protein is Wnt3& In certain embodiments the Wnt protein isco- or post-translationally modified as occurs when the Wnt protein isproduced in a host cell that naturally expresses the Wnt protein. Inother embodiments the Wnt protein is not co- or post-translationallymodified as in nature, in certain embodiments the soluble, biologicallyactive Wnt protein is modified with a lipid moiety such as palmitate.The lipid moiety may be attached to a conserved cysteine. For example,in certain embodiments the Wnt protein is palmitoylated on a conservedcysteine as known in the art. In certain embodiments the Wnt protein isglycosylated as occurs when the Wnt protein is produced in a mammalianhost cell that naturally expresses the Wnt protein. In other embodimentsthe Wnt protein is not glycosylated as found in nature. Recombinantmouse Wnt3a is commercially available (e.g., from Millipore cat. no.GF145 or R&D Systems cat no. 1324-WN-002).

In certain embodiments of the invention the Wnt pathway activator is anagent that increases the level of β-catenin, promotes its nuclearlocalization, or otherwise activates β-catenin signaling.

In certain embodiments of the invention the Wnt pathway activator is asmall molecule, by which is meant an organic compound having multiplecarbon-carbon bonds and a molecular weight of less than 1500 daltons.Typically such compounds comprise one or more functional groups thatmediate structural interactions with proteins, e.g., hydrogen bonding,and typically include at least an amine, carbonyl, hydroxyl or carboxylgroup, and in some embodiments at least two of the functional chemicalgroups. The small molecule agents may comprise cyclic carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more chemical functional groups and/orheteroatoms.

In certain embodiments of the invention the Wnt pathway activator is anagent that inhibits glycogen synthase kinase 3 (GSK3). These agentseffectively “turn on” the Wnt pathway without the need for extracellularWm. GSK3 is a serine/threonine kinase, originally identified as aregulator of glucose metabolism (reviewed in Frame and Cohen, Biochem J359:1-16, 2001; see also Cohen, Biochem Soc Trans 7:159-80, 1979; Embiet al., Bur J Biochem 107:519-27, 1980). “GSK3” as used herein refers toeither or both isoforms of GSK3(GSK3α and GSK3β). Inhibitors thatinhibit either or both of these isoforms are of use. In certainembodiments the GSK3 inhibitor specifically inhibits GSK3 and does notsubstantially inhibit the majority of other mammalian kinases. In someembodiments the GSK3 inhibitor does not substantially inhibit at least10 diverse mammalian kinases. In some embodiments the GSK3 inhibitorspecifically inhibits both GSK3β and GSK3α. In some embodiments the GSK3inhibitor specifically inhibits GSK3β but not GSK3β. For example, theIC50 for GSK3α may be at least 10-fold as great as for GSK3β. In someembodiments the GSK3 inhibitor specifically inhibits GSK3a but notGSK3α. For example, the IC50 for GSK3β may be at least 10-fold as greatas for GSK3α. In certain embodiments the IC50 of the GSK3 inhibitor forGSK3 is at least 10-fold lower than its IC50 for the majority of othermammalian kinases. In certain embodiments the IC50 of the GSK3 inhibitorfor GSK3 is less than 10 μM. In certain embodiments the IC50 of the GSK3inhibitor for GSK3 is less than 1 μM. It will be understood that theGSK3 inhibitor should be capable of entering cells in sufficientquantities under the conditions used so as to inhibit GSK3 therein. Insome embodiments the concentration of GSK3 inhibitor for used is atleast equal to the IC50 of the compound as measured in vitro. In someembodiments the concentration of GSK3 inhibitor used is no more than 100times the IC50 of the compound as measured in vitro. In some embodimentsthe concentration used ranges between 0.5 and 50-fold the IC50 of theagent as measured in vitro.

Many potent and selective small molecule inhibitors of GSK3 have nowbeen identified (Wagman A S, Johnson K W, Bussíere D E, Curr Pharm Des.,10(10): 1105-37, 2004). Exemplary GSK3 inhibitors of use include thefollowing: (1) BIO: (2′Z,3′E)-6-Bromoindirubin-3′-oxime,6-bromoindirubin-3-oxime (BIO) is a potent, reversible andATP-competitive GSK-3 inhibitor (Polychronopoulos, P. et al. J. Med.Chem. 47, 935-946, 2004). (2) AR-A014418:N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea. AR-A014418,inhibits GSK3 (IC50=104 nM), in an ATP-competitive manner (Ki=38 nM).AR-A014418 does not significantly inhibit cdk2 or cdk5 (IC50>100 μM) or26 other kinases, demonstrating high specificity for GSK3 (Bhat, R., etal., J. Biol. Chem. 278, 45937-45945, 2003). (3) SB 216763:3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione,See, e.g., Smith, D. G., et al, Bioorg. Med. Chem. Lett. 11, 635-639,(2001) and Cross, D. A., et al, J. Neurochem. 77, 94-102, (2001), (4) SB415286:3-[(3-Chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H)-pyrrol-2,5-dione.SB 415286 is described in Smith, D. G., et si, Bioorg. Med. Chem. Lett.11, 635-639, 2001 and Coughlan, M. P., et al, Chem. Biol. 10, 793-803,2000, TDZD-8: 4-Benzyl-2-methyl-2,4-thiadiazolidine-3,5-dione. Thiscompound is a selective inhibitor of GSK-3, a thiadiazolidinonederivative, a non-ATP competitive inhibitor of GSK-3β (IC50=2 μM). Itdoes not inhibit Cdk-1/cyclin B, CK-II, PKA or PKC at >100 μM. It hasbeen proposed to bind to the kinase site of GSK-30. (Martinez et al., J.Med. Chem. 45, 1292-1299, 2002); CHIR-911 and CHIR-837 (also retorted toas CT-99021 and CT-98023 respectively). Chiron Corporation (Emeryville,Calif.) and related compounds are of use. Lithium chloride, sodiumvalproate, and GSK3 inhibitor II (Calbiochem) are other GSK3 inhibitorsof use. Additional GSK3 inhibitors are described in U.S. Pat. Nos.6,057,117 and 6,608,063; U.S. patent application publications20040092535, 20040209878, 20050054663. Other GSK3 inhibitors of use aredescribed in WO/2003/049739, which discloses PYRIMIDINE COMPOUNDS USEFULAS GSK-3 INHIBITORS; WO/2002/085909, which discloses 9-DEAZAGUANINEDERIVATIVES AS INHIBITORS OF GSK-3, WO/2003/011287, which disclosesPYRAZOLON DERIVATIVES AS INHIBITORS OF GSK-3, WO/2005/039485, and/orWO/2006/091737.

In certain embodiments of the invention the Wnt pathway activator is acasein kinase 1 (CK1) inhibitor. Examples include D4476, IC261, andCKI-7 (see, e.g., Rena, G., et al. EMBO reports 5(1), 60-65, 2004).Compounds that inhibit CK1 and GSK3 are disclosed in U.S. Pat. No.7,098,204.

In certain embodiments of the invention the Wnt pathway activator is anactivator of a phosphatase that naturally dephosphorylates β-catenin atone or more of the sites phosphorylated by GSK3 or CK1.

The CREB binding protein (CBP) and the closely related protein p300 canassemble with β-catenin and act as β-catenin binding transcriptionalco-activators. For example, to generate a transcriptionally activecomplex, β-catenin recruits the transcriptional coactivators.CREB-binding protein (CBP) or its closely related homolog p300 (Hecht etal., EMBO J. 19:1839-50 (2000); Takemaru et al., J. Cell Biol.149:249-54 (2000)) well other component of the basel transcriptionmachinery. Other β-catenin co-activators include TBP, BRG1, BCL9/PYG,etc. The invention encompasses directly or indirectly modulating theinteractions between β-catenin and any one or more of theseco-activators so as to enhance the reprogramming of somatic cells. Forexample, the invention encompasses altering the relative participationof β-catenin in any one or mere of these complexes relative to itsparticipation in one or more other complexes. Agent such as smallmolecules may be used to selectively disrupt interaction of β-cateninwith a particular co-activator, thereby potentially reducingtranscription that would inhibit reprogramming or favor differentiation.Selective disruption may shift the balance towards interaction with adifferent co-activator to form a complex that enhances reprogramming.The agent may act directly on the complex or indirectly, e.g., bycausing post-translational modification such as phosphorylation ofβ-catenin or a co-activator. In one embodiment, the agent is a compounddescribed in U.S. Patent Pub. No. 20070128669 or an analog or derivativethereof, or an agent having the same mechanism of action. β-catenininteracting protein (also known as ICAT or CTNNBIP1) binds β-catenin andinhibits interaction between β-catenin and TCP family members (Gottardi,et al., Am J Physiol Cell Physiol. 286(4):C747-56, 2004). The encodedprotein is a negative regulator of the Wnt signaling pathway. Theinvention encompasses inhibiting ICAT (which tom includes any transcriptvariants or family members that inhibit the interaction of β-catenin andTCP) in order to activate the Wnt pathway. In certain embodiments of theinvention, the agent that activates a Wnt pathway does so by inhibitingexpression or activity of an endogenous inhibitor or negative regulatorof the Wnt pathway. In some embodiments, the agent inhibits expressionby RNA interference (RNAi). In some embodiments, the agent inhibitsexpression or activity of GSK3, ICAT, CK1, or CTNNBIP1.

In some embodiments an inhibitor of use in the present invention is anRNAi agent One of skill in the art will be able to identify anappropriate RNAi agent to inhibit expression of a gene of interest. See,e.g., Yu, J-Y., et at, Molecular Therapy, 7(2): 228-236, 2003. The RNAiagent may inhibit expression sufficiently to reduce the average steadystate level of the RNA transcribed from the gene (e.g., mRNA) or Itsencoded protein by, e.g., by at least 50%, 60%, 70%, 80%, 90%, 95%, ormono). The RNAi agent may contain a sequence between 17-29 nucleotideslong, e.g., 19-23 nucleotides long that is 100% complementary to themRNA or contains up to 1, 2, 3, 4, or 5 nucleotides, or up to about10-30% nucleotide, that do not participate in Watson-Crick base pairswhen aligned with the mRNA to achieve the maximum number ofcomplementary base pain. The RNAi agent may contain a duplex between17-29 nucleotide long in which all nucleotides participate inWatson-Crick base pairs or in which up to about 10-30% of thenucleotides do not participate in a Watson-Crick base pair. One of skillin the art will be aware of which sequence characteristics are oftenassociated with superior siRNA functionality and algorithms and rules bywhich such siRNAs can be designed (see, e.g., Jagla, B., et al, RNA,11(6):864-72, 2005). The methods of the invention can, but need not,employ siRNAs having such characteristics. In some embodiments, thesequence of either or both strands of the RNAi agent is/are chosen toavoid silencing non-target genes, e.g., the strand(s) may have less than70% 90%, or 90% complementarity to any mRNA other than the target mRNA.In some embodiments, multiple different sequences are used. Table 1lists the Gene IDs of the human and mouse genes encoding GSK3 and thenucleic acid (mRNA) and protein sequence accession numbers. RNAi agentscapable of silencing mammalian genes are commercially available (e.g.,from suppliers such as Qiagen, Dharmacon, Invitrogen, etc.). If multipleisoforms exist, one can design si RN As or shRNAs targeted against aregion present in all of the isoforms expressed in a given cell ofinterest.

Methods for silencing genes by transfecting cells with siRNA orconstructs encoding shRNA are known in the art. To express an RNAi agentin somatic cells, a nucleic acid construct comprising a sequence thatencodes the RNAi agent, operably linked to suitable expression controlelements, e.g., a promoter, can be introduced into the cells as known inthe art. For purposes of the present invention a nucleic acid constructthat comprises s sequence that encodes an RNA or polypeptide ofinterest, the sequence being operably linked to expression controlelements such as a promoter that direct transcription in a cell ofinterest, is referred to as an “expression cassette”, The promoter am bean RNA polymerase I, II, or III promoter functional in somatic mammaliancells. In certain embodiments, expression of the RNAi agent isconditional. In some embodiments, expression is regulated by placing thesequence that encodes the RNAi agent under control of a regulatable(e.g., inducible or repressible) promoter.

Constitutively active versions of proteins such as β-catenin or othercomponents of the Wnt signalling pathway are also of use. N-terminaltruncation or deletion of the potential GSK-3 phosphorylation site inthe N-terminal region or a missense mutation of the serine or threonineresidues therein results in the accumulation of truncated or normalsized β-catenin and then in activation of β-catenin-mediated signal (doLa Costa PNAS, 95(15): 8847-8851, 1998). Dominant negative versions ofendogenous proteins that inhibit Wnt signalling are also of use. In someembodiments, somatic cells are engineered to express these proteins. Insome embodiments, the protein is added to the culture medium.

In some embodiments, cells are treated to enhance uptake of a Wntpathway activator that acts intracellularly. For example, the cellmembrane may be partially permeabilized. In some embodiments, a Wntpathway activator is modified to comprise an amino acid sequence thatenhances cellular uptake of molecules by cells (also referred to as a“protein transduction domain”). Such uptake-enhancing amino acidsequences are found, e.g., in HIV-1 TAT protein, the herpes simplexvirus 1 (HSV-1) DNA-binding protein VP22, the Drosophila Antennapedia(Antp) transcription Actor, etc. Artificial sequences are also of use.See, e.g., Fischer et al, Bioconjugate Chem., Vol. 12, No. 6, 2001 andU.S. Pat. No. 6,835,810.

Without limitation, the invention contemplates use in the methods of thepresent invention of any of the compositions and approaches disclosed inU.S. Patent Pub. No. 20060147435 as being useful for promotingWnt/β-catenin signaling.

In some embodiments of the invention, somatic cells are treated so thatthey express a Wnt protein at levels greater than would be the casewithout such treatment. In some embodiments, somatic cells aregenetically engineered to stably or transiently express a Wnt protein atlevels greater titan would be the case without such treatment. In acmeembodiments of the invention somatic cells are treated so that theyexpress a Wnt pathway component such as β-catenin or a TCF/LEF at levelsgreater then would be the case without such treatment. In someembodiments of the invention, somatic cells are genetically engineeredto stably or transiently express a Wnt pathway component such asβ-catenin or a TCF/LEF at levels greater than would be the case withoutsuch treatment.

Methods of the invention may include treating the cells with multiplereprogramming agents either concurrently (i.e., during time periods thatoverlap at least in part) or sequentially and/or repeating the stops oftreating the cells with an agent. The agent used in the repeatingtreatment may be the same as, or different from, the one used during thefirst treatment. The cells may be contacted with a reprogramming agentfor varying periods of time. In some embodiments, the cells arecontacted with the agent for a period of time between 1 hour and 60days, e.g., between 10 and 30 days, e.g., for about 15-20 days.Reprogramming agents may be added each time the cell culture medium isreplaced. The reprogramming agents) may be removed prior to performing aselection to enrich for pluripotent cells or assessing the cells forpluripotency characteristics.

Reprogramming agents or candidate reprogramming agents of interestinclude a variety of compounds. Exemplary compounds include agents thatinhibit histone deacetylation, e.g., histone deacetylase (HDAC)inhibitors and agents that inhibit DMA methylation, e.g., DNAmethyltransferase inhibitors. Without wishing to be bound by theory, DNAdemethylation can regulate gene expression by “opening” the chromatinstructure detectable as increased nuclease sensitivity. This remodelingof chromatin structure allows transcription factors to bind to thepromoter regions, assembly of the transcription complex, and geneexpression.

The major classes of HDAC inhibitors include (a) Small chain fatty acids(e.g., valproic acid); (b) hydroxamate small molecule inhibitors (e.g.,SAHA and PXD101); (c) Non-hydroxamate small molecule inhibitors, e.g.,MS-275; and (d) Cyclic peptides; e.g., depsipeptide (ace, e.g., Carey Nand La Thangue N B, Curr Opin Pharmacol.; 6(4):369-75, 2006). Examplesof histone deacetylase inhibitors include Trichostatin A:[R-(E,E)]-7-[4-(Dimethylamino)phenyl]-N-hydroxy-4,6-dimethyl-7-oxo-2,4-heptadienamide,which inhibits histone deacetylase at nanomolar concentrations;resultant histone hyper-acetylation leads to chromatin relaxation rodmodulation of gene expression. (Yoshida, M., et al., Bioessays 17,423-430, 1995; Minucci, S., et al., Proc. Natl. Acid. Sci. USA 94,11295-11300, 1997; Brehm, A., et al., 1998; Medina, V., et al.,Induction of caspase-3 protease activity and apoptosis by butyrate andtrichostatin A (inhibitors of histone deacetylase): dependence onprotein synthesis and synergy with a mitochondrial/cytochromec-dependent pathway. Cancer Res. 57, 3097-3707, 1997; Kim, M. S., etal., Inhibition of histone deacetylase increases cytotoxicity to anticancer drugs targeting DNA. Cancer Res. 63, 7291-7300, 2003); Apicidin:Cyclo[(2S)-2-amino-8-oxodecanoyl-1-methoxy-L-tryptophyl-L-isoleucyl-(2R)-2-piperidinecarbonyl](Kwon, S. H., et al. J. Biol. Chem. 18, 2073, 2002; Han, J. W., et al.Cancer Res. 60, 6068, 2000; Colletti, S. L., et al. Bioorg. Med. Chem.11, 107, 2001; Kim J. S., et al. Biochem. Biophys. Res. Commun. 281,866, 2001).

A variety of DNA methylation inhibitors are known in the art and are ofuse in the invention. Sec, e.g., Lyko, F. and Brown, R., JNCI Journal ofthe National Cancer Institute, 97(20):1498-t 506, 2005. Inhibitors ofDNA methylation include nucleoside DNA methyltransferase inhibitors suchas decitabine (2′-deoxy-5-azacytidine), 5-azadeoxycytidine, andzebularine, non-nucleoside inhibitors such as the polyphenol(−)-epigallocatechin-3-gallate (EGCG) and the small molecule RG108(2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-3-(1H-indol-3-yl)propanoicacid), compounds described in WO2005085196 and phthalimides,succinimides and related compounds as described in WO2007007034. Threeadditional classes of compounds art: (1) 4-Aminobenzoic acidderivatives, such as the antiarrhythmic drug procainamide and the localanesthetic procaine; (2) the psammaplins, which also inhibits histonedeacetylase (Pina, I. C., J Org Chem., 68(10):3866-73, 2003); and (3)oligonucleotides, including siRNAs, shRNAi, and specific antisenseoligonucleotides, such as MG98. DNA methylation inhibitors may act by avariety of different mechanisms. The nucleoside inhibitors aremetabolized by cellular pathways before being incorporated into DNA.Alter incorporation, they function as suicide substrates for DNMTenzymes. The nonnucleoside inhibitors procaine,epigallocatechin-3-gallate (EGCG), and RG108 have been proposed toinhibit DNA methyhransferases by masking DNMT target sequences (i.e.,procaine) or by blocking the active site of the enzyme (i.e., EQCG andRG108). In some embodiments of the invention, combinations of DNAmethylation inhibitors are used. In some embodiments, the concentrationsare selected to minimize toxic effects on cells. In some embodimentsagents that incorporate into DNA (or whose metabolic productsincorporate into DNA) are not used.

DNA methyltransferase (DNMT1,3a, and/or 3b) and/or one or more HDACfamily members can alternatively or additionally be inhibited using RNAiagents.

The invention encompasses use of Wnt-conditioned medium, soluble Wnt orsmall molecules that modulate the Wnt signaling pathway in combinationwith other transient cues, e.g., small molecules, that can replace Oct4,Sox2, Klf4, Nanog, and/or Lin28 retroviruses in reprogramming somaticcells to pluripotency. The invention provides a composition comprising aWnt pathway modulator and at least one compound selected from the groupconsisting of: HDAC inhibitors and DNA methylation inhibitors. Theinvention provides a composition comprising a Wnt pathway modulator, atleast one HDAC inhibitor, and at least one DNA methylation inhibitor.The invention provides cell culture medium containing any of the abovecombinations of agents. In certain embodiments, the HDAC inhibitor isany HDAC inhibitor mentioned above. In certain embodiments, the DNAmethylation inhibitor is any HDAC inhibitor mentioned above. In certainembodiments, the Wnt pathway modulator activates the Wnt pathway. Incertain embodiments, the cell culture medium comprises Wnt-conditionedmedium, e.g., Wnt3a-CM, as the source of Wnt pathway modulator. Incertain embodiments, the Wnt pathway modulator is a small molecule. Incertain embodiments, the composition comprises somatic cells. In certainembodiments, the somatic cells are engineered to express at least one ofthe transcription factors Oct4, Nanog, Sox2, Klf4, and Lin28.

Somatic Cells and Reprogrammed Somatic Cells

Somatic cells of use the invention may be primary cells(non-immortalized cells), such as those freshly isolated from an animal,or may be derived from a cell line capable or prolonged proliferation inculture (e.g., for longer than 3 months) or indefinite proliferation(immortalised cells). Adult somatic cells may be obtained fromindividuals, e.g., human subjects, and cultured according to standardcell culture protocols available to those of ordinary skill w the art.The cells may be maintained in cell culture following their isolationfrom a subject. In certain embodiments, the cells are passaged once ormore following their isolation from the individual (e.g., between 2-5,5-10, 10-20, 20-50, 50-100 times, or mote) prior to their use in amethod of the invention. They may be frozen and subsequently thawedprior to use. In some embodiments, the cells will have been passaged nomore than 1, 2, 5, 10, 20, or 50 times following their isolation fromthe individual prior to their use in a method of the invention.

Somatic cells of use in the present invention include mammalian cells,such as, for example, human cells, non-human primate cells, or mousecells. They may be obtained by well-known methods from various organs,e.g., skin, lung, pancreas, liver, stomach, intestine, heart,reproductive organs, Madder, kidney, urethra and other urinary organs,etc., generally from any organ or tissue containing live somatic cells.Mammalian somatic cells useful in various embodiments of the presentinvention include, for example, fibroblasts, adult stem cells, sertolicells, granulosa cells, neurons, pancreatic islet cells, epidermalcells, epithelial cells, endothelial cells, hepatocytes, hair folliclecells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes,lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes,mononuclear cells, cardiac muscle cells, skeletal muscle cells, etc.,generally any living somatic cells.

Somatic cells may be treated so as to cause them to express or containone or mote reprogramming factor, pluripotency factor, and/orpluripotency inducing factor, at levels greeter then would be the casein the absence of such treatment. For example, somatic cells may begenetically engineered to express one or more genes encoding one or moresuch factor(s) and/or may be treated with agent(s) that increaseexpression of one or more endogenous genes encoding such factors and/orstabilize such factor(s). The agent could be, for example, a smallmolecule, a nucleic acid, a polypeptide, etc. In some embodiments,factors such as pluripotency factors are introduced into somatic cells,e.g., by microinjection or by contacting the cells with the factorsunder conditions in which the factors are taken up by the cells. In someembodiments, the factors are modified to incorporate a proteintransduction domain. In some embodiments, the cells are permeabilized orotherwise treated to increase their uptake of the factors, Exemplaryfactors are discussed below.

The transcription factor Oct4 (also called Pou5fl, Oct-3, Oct3/4) is enexample of a pluripotency factor. Oct4 has been shown to be required forestablishing and maintaining the undifferentiated phenotype of ES cellsand plays a major role in determining early events in embryogenesis andcellular differentiation (Nichols et al., 1998, Cell 95:379-391; Niwa etal., 2000, Nature Genet 24:372-376). Oct4 expression is down-regulatedas stem cells differentiate into more specialized cells. Nanog isanother example of a pluripotency factor. Nanog is a homeobox-containingtranscription factor with an essential function in maintaining thepluripotent cells of toe inner cell mass and in the derivation of EScells from these. Furthermore, overexpression of Nanog is capable ofmaintaining toe pluripotency and self-renewing characteristics of ESCsunder what normally would be differentiation-inducing cultureconditions. (See Chambers et al., 2003, Cell 113: 643-655; Mitsui etal., Cell. 2003, 113(5):631-42) Sox2, another pluripotency factor, is anHMO domain-containing transcription factor known to be essential fornormal pluripotent cell development and maintenance (Avilion, A., etal., Genes Dev. 17, 126-140, 2003). Klf4 is a Krüppel-type zinc fingertranscription (actor initially identified as a Klf family memberexpressed in the gut (Shields, J. M, et al., J. Biol. Chem.271:20009-20017, 1996). Overexpression of Klf4 in mouse ES cells wasfound to prevent differentiation in embryoid bodies formed in suspensionculture, suggesting that Klf4 contributes to ES self renewal (Li, Y., etal., Blood 105:635-637, 2005). Sox2 is a member of the family of SOX(sex determining region Y-box) transcription factors and is importantfor maintaining ES cell self-renewal. c-Myc is a transcription factorthat plays a myriad of roles in normal development and physiology aswell as being an oncogene whose dysregulated expression or mutation isimplicated in various types of cancer (reviewed in Pelengaris S, KhanM., Arch Biochem Biophys. 416(2):129-36, 2003; Cole M D, Nikiforov M A,Curr Top Microbiol Immunol., 302:33-50, 2006). In some embodiments, suchfactors are selected from: Oct4, Sox2, Klf4, and combinations thereof.In some embodiments, a different, functionally overlapping Klf familymember such as Klf2 is substituted for Klf4. In some embodiments, thefactors include at least Oct4. In some embodiments, the factors includeat least Oct4 and a Klf family member, e.g., Klf2. Lin28 is adevelopmentally regulated RNA binding protein. In some embodiments,somatic cells are treated so that they express or contain one or morereprogramming factors selected from: Oct4, Sox2, Klf4, Nanog, Lin28, andcombinations thereof. CCAAT/enhancer-binding-protein-alpha (C/EBPalpha)is another protein that promotes reprogramming at least in certain celltypes, e.g., lymphoid cells such as B-lineage cells, is considered areprogramming factor for such cell types, and b of use in certainembodiments of the invention, e.g., in combination with one or more ofthe pluripotency genes and/or Wnt pathway modulators described herein.

Other genes of interest are involved in chromatin remodeling and/or arehave been shown to be important for maintaining pluripotency of EScells. Optionally the gene is one that is downregulated as the cellsdifferentiate and/or is not expressed in adult somatic cells. Othergenes of interest encode microRNA precursors that have been associatedwith multipotency or pluripotency and/or dial are naturally expressed inmultipotent or pluripotent cells. Other genes of interest include encodeRNAi agents that Inhibit genes that are targets, of endogenous microRNAsthat are naturally expressed in multipotent or pluripotent cells.

In one embodiment, the exogenously introduced gene may be expressed froma chromosomal locus other than the chromosomal locus of an endogenousgene whose function is associated with pluripotency. Such a chromosomallocus may be a locus with open chromatin structure, and contain gene(s)whose expression is not required in somatic cells, e.g., the chromosomallocus contains gene(s) whose disruption will not cause cells to die.Exemplary chromosomal loci include, for example, the mouse ROSA 26 locusand type II collagen (Col2a1) locus (See Zambrowicz et al., 1997).

Methods for expressing genes in cells are known in the art Generally, asequence encoding a polypeptide or functional RNA such as an RNAi agentis operably linked to appropriate regulatory sequences. The termregulatory sequence includes promoters, enhancers and other expressioncontrol elements. Exemplary regulatory sequences are described inGoeddel; Gets Expression Technology: Methods in Enzymology, AcademicPress, San Diego, Calif. (1990). For instance, any of a wide variety ofexpression control sequences that control the expression of a DNAsequence when operatively linked to it may be used in these vectors toexpress cDNAs.

The exogenously introduced gene may be expressed from an inducible orrepressible regulatory sequence such that its expression can beregulated. The term “inducible regulatory sequence”, as used herein,refers to a regulatory sequence that, in the absence of an inducer (suchas a chemical and/or biological agent) or combination of inducers, doesnot direct expression, or directs low levels of expression of anoperably linked nucleic acid sequence such as a cDNA, and, in responseto an inducer, its ability to direct expression is enhanced. Exemplaryinducible promoters include, for example, promoters that respond toheavy metals (CRC Boca Raton, Fla. (1991), 167-220; Brinster et al.Nature (1982), 296, 39-42), to thermal shocks, to hormones (Lee et al.P.N.A.S. USA (1988), 85, 1204-1208; (1981), 294, 228-232; Klock et al.Nature (1987), 329, 734-736; Israel and Kaufman, Nucleic Adds Res.(1989), 17, 2589-2604), promoters that respond to chemical agents, suchas glucose, lactose, galactose or antibiotic. A “repressible regulatorysequence” is one that directs expression of an operably linked nucleicacid sequence in the absence of a specific agent or combination ofagents that inhibits expression.

A tetracycline-inducible promoter is an example of an inducible promoterthat responds to an antibiotic. See Gossen, M. and Bujard, H., Annu RevGenet. Vol. 36: 153-173 2002 and references therein. Thetetracycline-inducible promoter comprises a minimal promoter linkedoperably to one or more tetracycline operator(s). The presence oftetracycline or one of its analogues leads to the binding of atranscription activator to the tetracycline operator sequences, whichactivates the minimal promoter and hence the transcription of theassociated cDNA. Tetracycline analog includes any compound that displaysstructural similarity with tetracycline and is capable of activating atetracycline-inducible promoter. Exemplary tetracycline analogs include,for example, doxycycline, chlorotetracycline and anhydrotetracycline.

In some embodiments of the invention, expression of an introduced gene,e.g., a gene encoding a reprogramming factor or RNAi agent is transient.Transient expression can be achieved by transient transfection or byexpression from a regulatable promoter. In some embodiments, expressioncan be regulated by, or is dependent on, expression of a site-specificrecombinase. Recombinase systems include the Cre-Lox and Flp-Frtsystems, among others (Gossen, M. and Bujard, H., 2002). In someembodiments, a recombinase is used to him on expression by removing astopper sequence that would otherwise separate the coding sequence fromexpression control sequences. In some embodiments, a recombinase is usedto excise at least a portion of a gene after pluripotency has beeninduced. In some embodiments, the recombinase is expressed transiently,e.g., it becomes undetectable after about 1-2 days, 2-7 days, 1-2 weeks,etc. In some embodiments the recombinase is introduced from externalsources. Optionally the recombinase in these embodiments a proteintransduction domain.

Reprogrammed somatic cells may be assessed for one or mote pluripotencycharacteristic(s). The presence of pluripotency characteristic(s)indicates that the somatic cells have been reprogrammed to a pluripotentstate. The lean “pluripotency characteristics”, as used herein, refersto characteristics associated with and indicative of pluripotency,including, for example, the ability to differentiate into cells derivedfrom all three embryonic germ layers all types and a gene expressionpattern distinct for a pluripotent cell, including expression ofpluripotency factors and expression of other ES cell markers.

To assess potentially reprogrammed somatic cells for pluripotencycharacteristics, one may analyze such cells for particular growthcharacteristics and ES cell-like morphology. Cells may be injectedsubcutaneously into immunocompromised SC ID mice to determine whetherthey induce teratomas (a standard assay for ES cells). ES-like cells canbe differentiated into embryoid bodies (another ES specific feature).Moreover, ES-like cells can be differentiated in vitro by adding certaingrowth factors known to drive differentiation into specific cell types.Self-renewing opacity, marked by induction of telomerase activity, isanother pluripotency characteristic that can be monitored. One may carryout functional assays of the reprogrammed somatic cells by introducingthorn into blastocysts and determining whether the cells are capable ofgiving rise to all cell types. See Hogan et al., 2003. If thereprogrammed cells are capable of forming a few cell types of the body,they are multipotent; if the reprogrammed cells are capable of formingall cell types of the body including germ cells, they are pluripotent.

One may also examine the expression of an individual pluripotency lectorin the (reprogrammed somatic cells to assess their pluripotencycharacteristics. Additionally or alternately, one may assess theexpression of other ES cell markets. Stage-specific embryonic I 5antigens-1, -3, and -4 (SSEA-1, SSEA-3, SSEA-4) are glycoproteinsspecifically expressed in early embryonic development and are markersfor ES cells (Solter and Knowles, 1978, Proc. Natl. Acad. Sci. USA75:5565-5569; Kannagi et al., 1983, EMBO J 2:2355-2361). Elevatedexpression of the enzyme alkaline phosphatase (AP) is another markerassociated with undifferentiated embryonic stem cells (Wobus et al.1984, Exp. Cell 152:212-219; Pease et al., 1990, Dev. Biol.141:322-352). Additional ES cell markers are described in Ginis, I., etal., Dev. Biol., 269:369-380, 2004 and in The International Stem CellInitiative, Adewumi O, et al., Nat Biotech not., 25(7):803-16, 2007 andreferences therein. For example, TRA-1-60, TRA-1-81, GCTM2 and GCT343,and the protein antigens CD9, ThyI (also known as CD90), class I HLA,NANOG, TDGF1, DNMT3B, GABRB3 and GDF3, REX-1, TERT, UTF-1, TRF-1, TRF-2,connexin43, connexin45, Foxd3, FGFR-4, ABCG-2, and Glut-1 are of use.

One may perform expression profiling of the reprogrammed somatic cellsto assess their pluripotency characteristics. Pluripotent cells, such asembryonic stem cells, and multipotent cells, such as adult stem cells,are known to have a distinct pattern of global gene expression. See, forexample, Ramalho-Samos et al., Science 298:597-600, 2002; Ivanova etal., Science 298:601-604, 2002; Boyer, L A, et al. Nature 441, 349,2006, and Bernstein, B E, et al., Cell 125 (2), 315, 2006. One mayassess DNA methylation, gene expression, and/or epigenetic state ofcellular DNA, and/or developmental potential of the cells, e.g., asdescribed in Wernig, M., et al., Nature, 448:318-24, 2007. Celia thatare able to form teratomas containing cells having characteristics ofendoderm, mesoderm, and ectoderm when injected into SCID mice and/orpossess ability to participate (following injection into murineblastocysts) in formation of chimeras that survive to term areconsidered pluripotent Another method of use to assess pluripotency isdetermining whether the cells have reactivated a silent X chromosome.

Somatic cells may be reprogrammed to gain either a complete set of thepluripotency characteristics. Alternatively, somatic cells may bereprogrammed to gain only a subset of the pluripotency characteristics.

Certain methods of the invention include a step of selecting cells thatexpress a marker that is expressed by multi potent or pluripotent cells.The marker may be sped Really expressed in such cells. Standard cellseparation methods, e.g., flow cytometry, affinity separation, etc maybe used. Alternately or additionally, one could select cells that do notexpress markers characteristic of somatic cells from which thepotentially reprogrammed cells were derived and which are not expressedin ES cells generated using conventional methods. Other methods ofseparating cells may utilize differences in average cell size or densitythat may exist between in impotent cells and somatic cells. For example,cells can be filtered through materials having pores that will allowonly certain cells to pass through.

In some embodiments, the somatic cells contain a nucleic acid comprisingregulatory sequences of a gene encoding a pluripotency factor operablylinked to a selectable or detectable marker (e.g., GPP or neo). Thenucleic acid sequence encoding the marker may be integrated at theendogenous locus of the gene encoding the pluripotency factor (e.g.,Oct4) or the construct may comprise regulatory sequences operably linkedto the marker. Expression of the marker may be used to select, identify,and/or quantify reprogrammed cells.

Any of the methods of the invention that relate to generating areprogrammed somatic cell may include a step of obtaining a somatic cellor obtaining a population of somatic cells from an individual in need ofcell therapy. Reprogrammed somatic cells are generated, selected, oridentified from among the obtained cells or cells descended from theobtained cells. Optionally the cell(s) are expended in culture prior togenerating, selecting, or identifying reprogrammed somatic cell(s)genetically matched to the donor.

In some embodiments colonies are subcloned and/or passaged once or morein order to obtain a population of cells enriched for ES-like cells. Theenriched population may contain at least 95%, 96%, 97%, 98%, 99% ormore, e.g., 100% ES-like cells. The invention provides cell Knee ofsomatic cells that have been stably and heritably reprogrammed to anES-like state.

In some embodiments, the methods are practiced using somatic cells thatare not genetically engineered for purposes of identifying or selectingreprogrammed cells. The resulting reprogrammed somatic cells do notcontain exogenous genetic material that has been introduced into saidcells (or ancestors of said cells) by the hand of man, e.g., forpurposes of identifying or selecting reprogrammed cells. In someembodiments, the somatic cells and reprogrammed somatic cells derivedtherefrom do contain exogenous genetic material in their genome, butsuch genetic material is introduced for purposes of correcting a geneticdefect in such cells or enabling such cells to synthesize a desiredprotein for therapeutic purposes and is not used to identify or selectreprogrammed cells.

In some embodiments, the methods employ morphological criteria toidentify reprogrammed somatic cells from among a population of somaticcells that are not reprogrammed. In some embodiments, the methods employmorphological criteria to identify somatic cells that have beenreprogrammed to an ES-like state from among a population of cells thatare not reprogrammed or are only pertly reprogrammed to an ES-likestate. “Morphological criteria” is used in a broad sense to refer to anyvisually detectable feature or characteristic of the cells or colonies.Morphological criteria include, e.g., the shape of the colonies, thesharpness of colony boundaries, the density, small size, and roundedshape of the cells relative to non-reprogrammed cells, etc. FIG. 1 showscolonies of cells displaying morphological criteria indicative of cellsthat have been reprogrammed to an ES-like state. Note the dense coloniescomposed of small, rounded cells, and the sharp colony boundaries. Theinvention encompasses identifying and, optionally, isolating colonies(or cells from colonies) wherein the colonies display one or more suchcharacteristics. The reprogrammed somatic cells may be identified ascolonies growing in a first cell culture dish (which term refers to anyvessel, plate, dish, receptacle, container, etc., in which living cellscan be maintained in vitro) and the colonies, or portions thereof,transferred to a second cell culture dish, thereby isolatingreprogrammed somatic cells. Hie cells may then be further expended.

Methods of Screening for an Agent that Reprograms or Contributes toReprogramming Somatic Cells

The present invention also provides methods for identifying an agentthat, alone or in combination with one or more other agents, reprogramssomatic cells to a less differentiated state. The invention furtherprovides agents identified according to the methods, in one embodiment,the methods comprise contacting somatic cells with a Wnt pathwayactivator and a candidate agent and determining whether the presence ofthe candidate agent results in enhanced reprogramming (e.g., increasedreprogramming speed and/or efficiency) relative to that which wouldoccur if cells had not been contacted with the candidate agent. In someembodiments, the Wnt activator and candidate agent are present togetherin the cell culture medium while in other embodiments the Wnt activatorand the candidate agent are not present together (e.g., the cells areexposed to the agents sequentially). The cells may be maintained inculture liar, e.g., at least 3 days, at least 5 days, up to 10 days, upto 15 days, up to 30 days, etc., during which time they are contactedwith the Wnt activator and the candidate agent for all or pert of thetime. In some embodiments, the agent is identified as an agent thatreprograms cells if there are at least 2, 5, or 10 times as manyreprogrammed cells or colonies comprising predominantly reprogrammedcells after said time period than if the cells have not been contactedwith the agent.

A candidate agent can be any molecule or supramolecular complex, e.g. apolypeptide, peptide (which is used herein to refer to a polypeptidecontaining 60 amino acids or less), small organic or inorganic molecule(i.e., molecules having a molecular weight less than 1,500 Da, 1000 Da,or 500 Da), polysaccharide, polynucleotide, etc. which is to be testedfor ability to reprogram cells. In some embodiments, the candidateagents are organic molecules, particularly small organic molecules,comprising functional groups that mediate structural interactions withproteins, e.g., hydrogen bonding, and typically include at least anamine, carbonyl, hydroxyl or carboxyl group, and in some embodiments atleast two of the functional chemical groups. The candidate agents maycomprise cyclic carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more chemical functionalgroups and/or heteroatoms.

Candidate agents are obtained from a wide variety of sources, as will beappreciated by those in the art, including libraries of synthetic ornatural compounds. In some embodiments, candidate agents are syntheticcompounds. Numerous techniques are available for the random and directedsynthesis of a wide variety of organic compounds and biomolecules. Insome embodiments, the candidate modulators are provided as mixtures ofnatural compounds in the form of bacterial, fungal, plant and animalextracts, fermentation broths, conditioned media, etc., that areavailable or readily produced.

In some embodiments, a library of compounds is screened. A library istypically a collection of compounds that can be presented or displayedsuch that the compounds can be identified in a screening assay. In someembodiments, compounds in the library are housed in individual wells(e.g., of microtiter plates), vessels, tubes, etc., to facilitateconvenient transfer to individual wells or vessels for contacting cells,performing cell-free assays, etc. The library may be composed ofmolecules having common structural features which differ in the numberor type of group attached to the main structure or may be completelyrandom. Libraries include but are not limited to, for example, phagedisplay libraries, peptide libraries, polysome libraries, aptamerlibraries, synthetic small molecule libraries, natural compoundlibraries, and chemical libraries. Methods for preparing libraries ofmolecules are well known in the art and many libraries are availablefrom commercial or non-commercial sources. Libraries of interest includesynthetic organic combinatorial libraries. Libraries, such as, syntheticsmall molecule libraries and chemical libraries can comprise astructurally diverse collection of chemical molecules. Small moleculesinclude organic molecules often having multiple carbon-carbon bonds. Thelibraries can comprise cyclic carbon or heterocyclic structure and/oraromatic or polyaromatic structures substituted with one or morefunctional groups. In some embodiments, the small molecule has between 5and 50 carbon atoms, e.g., between 7 and 30 carbons. In someembodiments, the compounds are macrocyclic. Libraries of interest alsoinclude peptide libraries, randomized oligonucleotide libraries, and thelike. Libraries can be synthesized of peptoids and non-peptide syntheticmoieties. Such libraries can further be synthesised which containnon-peptide synthetic moieties which are less subject to enzymaticdegradation compared so their naturally occurring counterparts, Smallmolecule combinatorial libraries may also be generated. A combinatoriallibrary of small organic compounds may comprise a collection of closelyrelated analogs dial differ from each other in one or more points ofdiversity and are synthesized by organic techniques using multi-stepprocesses. Combinatorial libraries can include a vast number of smallorganic compounds. A “compound array” as used herein is a collection ofcompounds identifiable by their spatial addresses in Cartesiancoordinates and arranged such that each compound has a common molecularcore and one or more variable structural diversity elements. Thecompounds in such a compound array are produced in parallel in separatereaction vessels, with each compound identified and tracked by itsspatial address. Examples of parallel synthesis mixtures and parallelsynthesis methods are provided in U.S. Pat. No. 5,712,171. In someembodiments, mixtures containing two or mote compounds, extracts orother preparations obtained from natural sources (which may comprisedozens of compounds or more), and/or inorganic compounds, eta., arescreened.

In one embodiment, the methods of the invention are used to screen“approved drugs”. An “approved drug” is any compound (which termincludes biological molecules such as proteins and nucleic acids) whichhas been approved for use in humans by the FDA or a similar governmentagency in another country, for any purpose. This can be a particularlyuseful class of compounds to screen because it represents a set ofcompounds which are believed to be safe and, at least in the case of FDAapproved dregs, therapeutic for at least one purpose. Thus, there is ahigh likelihood that these drugs will at least be safe for otherpurposes.

Representative examples of libraries that could be screened includeDIVERSet™, available from ChemBridge Corporation, 16981 Via Tazon, SanDiego, Calif. 92127. DIVERSet contains between 10,000 and 50,000drug-like, hand-synthesized small molecules. The compounds arepre-selected to form a “universal” library that covers the maximumpharmacophore diversity with the minimum number of compounds and issuitable for either high throughput or lower throughput screening. Fordescription of additional libraries, see, for example, Tan, et al., Am.Chem Soc. 120, 8565-8566, 1998; Floyd C D, Leblanc C, Whittaker M, ProgMed Chem 36:91-168, 1999. Numerous libraries are commercially available.e.g., from AnalytiCon USA Inc., P.O. Box 5926, Kingwood, Tex. 77325;3-Dimensional Pharmaceuticals, Inc., 665 Stockton Drive, Suite 104,Extern, Pa. 19341-1151; Tripos, Inc., 1699 Hanky Rd., St Louis, Mo.,63144-2913, etc. For example, libraries based on quinic acid andshikimic acid, hydroxyproline, santonine, dianhydro-D-glucitol,hydroxypipecolinic acid, andrographolide, piperazine-2-carboxylic acidbased library, cytosine, etc., are commercially available.

In some embodiments, the candidate agents are cDNAs from a cDNAexpression library prepared from cells, e.g., pluripotent cells. Suchcells may be embryonic stem cells, oocytes, blastomeres,teratocarcinomas, embryonic germ cells, inner cell mess cells, etc.

It will be appreciated that the candidate reprogramming agent to betested is typically one that is not present in standard culture medium,or if present is present in lower amounts than when used in the presentinvention.

It will also be appreciated that a useful reprogramming agent or otherform of reprogramming treatment need not be capable of reprogramming alltypes of somatic cells and need not be capable of reprogramming allsomatic cells of a given cell type. Without limitation, a candidateagent that results in a population that is enriched for reprogrammedcells by a factor of 2, 5, 10, 50, 100 or more (i.e., the fraction ofreprogrammed cells in the population is 2, 5, 10, 50, or 100 times morethan present in a starting population of cells heated in the same waybut without being contacted with the candidate agent) is of use.

In some embodiments of the invention, the inventive screening method isused to identify an agent or combination of agents that substitutes forKlf4 in reprogramming cells to an ES-tike state. The method may bepracticed using somatic cells engineered to express Sox2 and Oct4 andcontacted with a Wnt pathway activator. In some embodiments, the methodis used to identity an agent that substitutes for Sox2 in reprogrammingcells to an BS-like state. The method may be practiced using somaticcells engineered to express Klf4 and Oct4 and contacted with a Wntpathway activator. In some embodiments, the method is used to identifyan agent that substitutes for Oct4 in reprogramming cells to an ES-likestate. The method may be practiced using somatic cells engineered toexpress Sox2 and Klf 4 and contacted with a Wnt pathway activator. It iscontemplated that engineered expression of Klf4, Sox2, Oct4, and c-Mycis replaced by treating somatic cells with a combination of smallmolecules and/or polypeptides or other agents that do not involvemodification of the genome. In some embodiments, the methods arepracticed using human cells. In some embodiments, the methods arepracticed using mouse cells. In some embodiments, the methods arepracticed using non-human primate cells.

The invention encompasses testing Wnt pathway modulators, e.g.,libraries of small molecules known or suspected to modulate the Wntpathway, to identity those that are effective in enhancing reprogrammingand/or have superior ability to enhance reprogramming somatic cells topluripotency, e.g., relative to other compounds tested. In someembodiments, at least 10, at least 20, at least 50, at least 100, or atleast 1,000 small molecules, e.g., structurally related molecules, atleast some of which are known or believed to modulate Wnt pathwayactivity, are tested. In some embodiments, a Wnt inhibitor is used toconfirm that a compound that enhances reprogramming and is suspected ofdoing so by modulating Wnt pathway activity docs in feet act via the Wntpathway. For example, if the Wnt pathway inhibitor blocks the effect ofa test compound on reprogramming, it may be concluded feat tire testcompound acts via the Wnt pathway.

The methods and compositions of the present invention relating to Wntpathway modulation may be applied to or used in combination with variousother methods and compositions useful for somatic cell reprogrammingand/or for identifying reprogramming agents for use in somatic cellreprogramming. Such combined methods and compositions are aspects of theinvention. For example, some embodiments of the invention employ celltypes (e.g., neural stem cells or progenitor cells) that naturallyexpress one or more reprogramming factors at levels higher than suchfactor(s) are expressed in many other cell types (see, e.g., Eminli, etal., Reprogramming of Neural Progenitor Cells into iPS Cells in theAbsence of Exogenous Sox2 Expression, Stem Cells. 2008 Jut 17, epubahead of print).

The methods and compositions may be used together with methods andcompositions disclosed in PCT/US2008/004516, which is incorporatedheroin by reference:

Genetically homogeneous ‘secondary’ somatic cells that carryreprogramming fedora as defined doxycycline (dox)-inducible transgeneshave been derived Wenrig, et al., A novel drug-inducible transgenicsystem for direct reprogramming of multiple somatic cell types. NatureBiotechnology; published online 1 Jul. 2008; doi:10.1038/nbt1483). Thesecells were produced by infecting fibroblasts with dox-induciblelentiviruses, reprogramming by dox addition, selecting inducedpluripotent stem cells and producing chimeric mice. Cells derived fromthese chimeras reprogram upon dox exposure without the need for viralinfection with efficiencies 25- to 50-fold greater than those observedusing direct infection and drug selection for pluripotency markerreactivation. In some embodiments of the invention, such secondarysomatic cells are used in embodiments of the present invention and/orsecondary somatic cells are generated without use of c-Myc virus byemploying Wnt pathway stimulation as described herein. The instantinvention contemplates use of Wnt pathway modulation in compositions andmethods relating to secondary somatic cells.

In some embodiments of the invention, the somatic cells contain anucleic acid sequence encoding a selectable marker, operably linked to apromoter of an endogenous pluripotency gene, e.g., Oct4 or Nanog. Thesequence encoding the marker may be integrated into the genome at theendogenous locus. The selectable marker may be, e.g., a readilydetectable protein such as a fluorescent protein, e.g., GPP or aderivative thereof. Expression of the marker is indicative ofreprogramming and can thus be used to identify or select reprogrammedcells, quantify reprogramming efficiency, and/or to identify,characterize, or use agents that enhance reprogramming and/or are beingtested for their ability to enhance reprogramming.

Reprogrammed Somatic Cells and Uses Thereof

The present invention provides reprogrammed somatic cells (RSCs),including induced pluripotent stem cells (iPS cells), produced by themethods of the invention. Those cells have numerous applications inmedicine, agriculture, and other areas of interest, some of which aredescribed here.

The invention provides methods for the treatment or prevention of scondition in a mammal. In one embodiment, the methods involve obtainingsomatic cells from the individual, reprogramming the somatic cells soobtained by methods of the present invention to obtain RSCs, e.g., iPScells. The RSCs are then cultured under conditions suitable for theirdevelopment into cells of a desired cell type. The developed cells ofthe desired cell type are introduced into the individual to treat thecondition. In an alternative embodiment, the methods start withobtaining somatic cells from the individual, reprogramming the somaticcells so obtained by methods of the present invention. The RPCs are thencultured under conditions suitable for development of the RPCs into adesired organ, which is harvested and introduced into the individual totreat the condition. The condition may be any condition in which cell ororgan function is abnormal and/or reduced below normal levels. Thus, theinvention encompasses obtaining somatic cells from an individual in needof cell therapy, reprogramming the cells by a process that comprisesactivating a Wnt pathway and/or culturing the cells in Wnt conditionedmedium, optionally differentiating reprogrammed somatic cells them togenerate cells of one or more desired cell types, and introducing thecells into the individual. An individual m need of cell therapy maysuffer from any condition, wherein the condition or one or more symptomsof the condition can be alleviated by administering cells to the donorand/or in which the progression of the condition can be slowed byadministering cells to the individual. The method may include a step ofidentifying or selecting reprogrammed somatic cells and separating themfrom cells that are not reprogrammed.

The RSCs in certain embodiments of the present invention are ES-likecells, also referred to as iPS cells, and thus may be induced todifferentiate to obtain the desired cell types according to knownmethods to differentiate ES cells, for example, the iPS cells may beinduced to differentiate into hematopoietic stem cells, muscle cells,cardiac muscle cells, liver cells, pancreatic cells, cartilage cells,epithelial cells, urinary tract cells, nervous system cells (e.g.,neurons) etc., by culturing such cells in differentiation medium andunder conditions which provide for edit differentiation. Medium sodmethods which result in the differentiation of embryonic stem cellsobtained using traditional methods are known in the art, as are suitableculturing conditions. Such methods and culture conditions may be appliedto the iPS cells obtained according to the present invention. See, e.g.,Trounson, A., The production and directed differentiation of humanembryonic stem cells, Endocr Rev. 27(2):206-19, 2006 and referencestherein, all of which are incorporated by reference, for some examples.See also Yao, S., et al, Long-term self-renewal and directeddifferentiation of human embryonic stem cells in chemically definedconditions, Proc Natl Acad. Sci USA, 103(18): 6907-6912, 2006 andreferences therein, all of which ere incorporated by reference.

Thus, using known methods and culture medium, one skilled in the sit mayculture the reprogrammed pluripotent cells to obtain desireddifferentiated cell types, e.g., neural cells, muscle cells,hematopoietic cells, etc. The subject cells may be used to obtain anydesired differentiated cell type. Such differentiated human cells afforda multitude of therapeutic opportunities. For example, humanhematopoietic stem cells derived from cells reprogrammed according tothe present invention may be used in medical treatments requiring bonemarrow transplantation. Such procedures are used to treat many diseases,e.g., late stage cancers and malignancies such as leukemia. Such cellsare also of use to treat anemia, diseases that compromise the immunesystem such as AIDS, etc. The methods of the present invention can alsobe used to treat, prevent, or stabilise a neurological disease such asAlzheimer's disease, Parkinson's disease, Huntington's disease, or ALS,lysosomal storage diseases, multiple sclerosis, or a spinal cord injury.For example, somatic cells may be obtained from the individual in needof treatment, and reprogrammed to gain pluripotency, and cultured toderive neurectoderm cells that may be used to replace or assist thenormal function of diseased or damaged tissue.

Reprogrammed cells that produce a growth factor or hormone such asinsulin, etc., may be administered to a mammal for the treatment orprevention of endocrine disorders. Reprogrammed epithelial cells may beadministered to repair damage to the lining of a body cavity or organ,such as a lung, gut, exocrine gland, or urogenital tract. It is alsocontemplated that reprogrammed cells may be administered to a mammal totreat damage or deficiency of cells in an organ such as the bladder,brain, esophagus, fallopian tube, heart, intestines, gallbladder,kidney, liver, lung, ovaries, pancreas, prostate, spinal cord, spleen,stomach, testes, thymus, thyroid, trachea, ureter, urethra, or uterus.

The present invention has the potential to provide an essentiallyunlimited supply of genetically matched cells suitable fortransplantation. Such a supply would address the significant problemassociated with current transplantation methods, i.e., rejection of thetransplanted tissue which may occur because of host versus graft orgraft versus host rejection. RSCs may also be combined with a matrix toform a tissue or organ in vitro or in vivo that may be used to repair orreplace a tissue or organ in a recipient mammal. For example, RSCs maybe cultured in vitro in the presence of a matrix to produce a tissue ororgan of the urogenital, cardiovascular, or musculoskeletal system.Alternatively, a mixture of the cells and a matrix may be administeredto a mammal for the formation of the desired tissue in vivo. The RSCsproduced according to the invention may be used to produce geneticallyengineered or transgenic differentiated cells, e.g., by introducing adesired gene or genes, or removing all or part of an endogenous gene orgenes of RSCs produced according to the invention, and allowing suchcells to differentiate into the desired cell type. One method forachieving such modification is by homologous recombination, whichtechnique can be used to insert, delete or modify a gene or genes at aspecific site or sites in the genome.

This methodology can be used to replace defective genes or to introducegenes which result in the expression of therapeutically beneficialproteins such as growth factors, hormones, lymphokines, cytokines,enzymes, etc. For example, the gene encoding brain derived growth factormay be introduced into human embryonic or stem-like cells, the cellsdifferentiated into neural cells and the cells transplanted into aParkinson's patient to retard the loss of neural cells during suchdisease. Using known methods to introduced desired genes/mutations intoES cells, RSCs may be genetically engineered, and the resultingengineered cells differentiated into desired cell types, e.g.,hematopoietic cells, neural cells, pancreatic cells, cartilage cells,etc. Genes which may be introduced into the RSCs include, for example,epidermal growth factor, basic fibroblast growth factor, glial derivedneurotrophic growth factor, insulin-like growth factor (I and II),neurotrophin3, neurotrophin-4/5, ciliary neurotrophic factor, AFT-1,cytokine genes (interleukins, interferons, colony stimulating factors,tumor necrosis factors (alpha and beta), etc.), genes encodingtherapeutic enzymes, collagen, human serum albumin, etc.

Negative selection systems known in the art can be used for eliminatingtherapeutic cells from a patient if desired. For example, cellstransfected with the thymidine kinase (TK) gene will lead to theproduction of embryonic (e.g., ES-like) cells containing the TK gene.Differentiation of these cells will lead to the isolation of therapeuticcells of interest which also express the TK gene. Such cells may beselectively eliminated at any time from a patient upon gancycloviradministration. Such a negative selection system is described in U.S.Pat. No. 5,698,446. In other embodiments the cells are engineered tocontain a gene that encodes a toxic product whose expression is undercontrol of an inducible promoter. Administration of the inducer causesproduction of the toxic product, leading to death of the cells. Thus anyof the somatic cells of the invention may comprise a suicide gene,optionally contained in an expression cassette, which may be integratedinto the genome. The suicide gene is one whose expression would belethal to cells. Examples include genes encoding diphtheria toxin,cholera toxin, ricin, etc. The suicide gene may be under control ofexpression control elements that do not direct expression under normalcircumstances in the absence of a specific inducing agent or stimulus.However, expression can be induced under appropriate conditions, e.g.(i) by administering an appropriate inducing agent to a cell or organismor (ii) if a particular gene (e.g., an oncogene, a gene involved in thecell division cycle, or a gene indicative of dedifferentiation or lossof differentiation) is expressed in the cells, or (iii) if expression ofa gene such as a cell cycle control gene or a gene indicative ofdifferentiation is lost. See, e.g., U.S. Pat. No. 6,761,884. In someembodiments the gene is only expressed following a recombination eventmediated by a site-specific recombinase. Such an event may bring thecoifing sequence into operable association with expression controlelements such as a promoter. Expression of the suicide gene may beinduced if it is desired to eliminate cells (or their progeny) from thebody of a subject after the cells (or their ancestors) have beenadministered to a subject. For example, if a reprogrammed somatic cellgives rise to a tumor, the tumor can be eliminated by inducingexpression of the suicide gene. In some embodiments tumor formation isinhibited because the cells are automatically eliminated upondedifferentiation or lose of proper cell cycle control.

Examples of diseases, disorders, or conditions that may be treated orprevented include neurological, endocrine, structural, skeletal,vascular, urinary, digestive, integumentary, blood, immune, auto-immune,inflammatory, endocrine, kidney, bladder, cardiovascular, cancer,circulatory, digestive, hematopoietic, and muscular diseases, disorders,and conditions. In addition, reprogrammed cells may be used forreconstructive application, such as for repairing or replacing tissuesor organs. In some embodiments, it may be advantageous to include growthfactors and proteins or other agents that promote angiogenesis.Alternatively, the formation of tissues can be effected totally invitro, with appropriate culture media and conditions, growth factors,and biodegradable polymer matrices.

With respect to the therapeutic methods of the invention theadministration of RSCs to a mammal is not limited to a particular modeof administration, dosage, or frequency of dosing; the present inventioncontemplates all modes of administration, including intramuscular,intravenous, intraarticular, intralesional, subcutaneous, or any otherroute sufficient to provide a dose adequate to prevent or treat adisease. The RSCs may be administered to the mammal in a single dose ormultiple doses. When multiple doses are administered, the doses may beseparated from one another by, for example, one week, one month, oneyear, or ten years. One or more growth factors, hormones, interleukins,cytokines, or other cells may also be administered before, during, orafter administration of the cells to further bias them towards aparticular cell type.

The RSCs of the present invention may be used as an in vitro model ofdifferentiation, in particular for the study of genes which are involvedin the regulation of early development. Differentiated cell tissues andorgans generated using the reprogrammed cells may be used to studyeffects of drugs and/or identify potentially useful pharmaceuticalagents.

Further Applications of Somatic Cell Reprogramming Methods andReprogrammed Cells

The reprogramming methods disclosed herein may be used to generate RSCs,e.g., iPS cells, for a variety of animal species. The RSCs generated canbe useful to produce desired animals. Animals include, for example,avians and mammals as well as any animal that is an endangered species.Exemplary birds include domesticated birds (e.g., quail, chickens,ducks, geese, turkeys, and guinea hens). Exemplary mammals includemurine, caprine, ovine, bovine, porcine, canine, feline and non-humanprimate. Of these, preferred members include domesticated animals,including, for examples, cattle, pigs, horses, cows, rabbits, guineapigs, sheep, and goats.

Methods for Gene Identification

The invention provides methods for identifying a gene whose expressioninhibits generation of reprogrammed cells. One method comprises: (i)activating the Wnt pathway in somatic cells; (ii) reducing expression ofa candidate gene by RNAi; (iii) determining whether reducing expressionof the candidate gene results in Increased efficiency of reprogrammingand, if so, identifying the candidate gene as one whose expressioninhibits reprogramming of somatic cells. One method comprises: (i)culturing somatic cells in Wnt conditioned medium; (ii) reducingexpression of a candidate gene by RNAi; (iii) determining whetherreducing expression of the candidate gene results in increasedefficiency of reprogramming and, if so, identifying the candidate geneas one whose expression inhibits reprogramming of somatic cells.Optionally the somatic cells are engineered to express at least one geneselected from: Oct4, Sox2, Nanog, Lin28, and Klf4. Optionally the cellsare contacted with Wnt pathway modulator. Libraries of shRNA or siRNA ofuse m the method are commercially available. The identified gene is atarget for inhibition in order to enhance cellular reprogramming. Agentsthat inhibit the gene (either RNAi agents or other agents such as smallmolecules) are of use to reprogram somatic cells, e.g., in conjunctionwith a Wnt activator.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following example, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Materials and Methods for Example 1

Cell Culture, Viral Infections, Induction of Gene Expression.

Cells wore cultured in 15% FBS, DMEM-KO, Penn/Step, Glutamine,Nonessential amino acids, β-ME, and LIF. Mouse embryo fibroblasts (MEFs)with an Oct4-IRES-eGFP construct (Meissner, A., et al., NatureBiotechnology, Direct reprogramming of genetically unmodifiedfibroblasts into pluripotent stem cells. Published online: 27 Aug.2007|dot:10.1038/nbt1335) inserted into the endogenous Oct4 locus wereinfected with lentiviral vectors driving fee doxycycline-inducibleexpression of Oct4, Sox2, Klf4 and c-Myc or only Oct4, Sox2, and Klf4.The vectors were based on the FUGW lentiviral vector backbone (Lois C,et al., Science 2002; 295:868-872), modified to include a tet-induciblepromoter. Two days following infliction cells were split and inducedwith doxycycline in the presence or absence of Wnt3a conditioned media(used in a 1:1 dilution with normal ES media with 2×LIF). These cellswere monitored for GFP expression by flow cytometry at day 13 and againat day 20. In parallel, MEFs with doxycycline-inducible Oct4 expressedfrom the collagen locus and Oct4-IRES-(neo resistance) inserted into theendogenous Oct4 locus were infected wife lentiviruses driving theoverexpression of either Sox2, Klf4 and c-Myc or Sox2 and Klf4. Again,two days following infection cells were split and induced withdoxycycline in the presence or absence of Wnt3a conditioned media.Separate plates of these cells were selected wife G418 at day 7 and day13 respectively. Following at least one week of G418 selection,resistant colonies were examined and counted.

Conditioned Medium.

Wnt3a conditioned media (CM) was collected from mouse L cells feat hadbeen transfected wife Wnt3a cDNA (Shibamoto et al. 1998). These cellsare available through ATCC (CRL-2647) along with the untransfectedparent cell line (CRL-2648) to use for control conditioned medium. Wnt3atransfected cells secrete Wnt, reaching levels up to 400 ng/mL of theWnt3a protein in their growth media. The basal medium consisted of DMEM,15% FBS, Penn/Strep, Glutamine and nonessential amino acids, preparedaccording the protocol of Single et al. (Single, et al., Biochem BiophysRes Common., 345(2):789-95, 2006). The media collected from thesecreting fibroblasts was filtered and diluted 1:1 wife regular ES cellmedia (15% FBS, DMEM-KO, Penn/Step, Glutamine, Nonessential amino acids,β-ME, and LIF). This media was then used to treat ES cells. The Wnt3aconditioned media has been shown by Applicants and others to activatethe Wnt signaling pathway in ES cells, as demonstrated by immunoblotsexamining beta-catenin phosphorylation.

Example 1: Generation of ES-Like Cells Using Wnt3a Conditioned Media

We hypothesized that stimulation of the stimulation of the Wnt pathwayusing soluble factors could modulate the efficiency of inducingpluripotency in somatic cells. This Example describes initialexperiments undertaken to determine the effect of Wnt pathwaystimulation on reprogramming. Cells containing an Oct4-IRES-eGFP orOct4-IRES-neo construct were infected with lend viral vectors encodingeither three or four factors as described above. Expression of thepluripotency factors was induced on day 2. In some experiments, cellswere cultured in Wnt3a conditioned media or unconditioned media as shownin FIG. 4A (top) from days 2-13. GFP expression was analyzed by FACS onday 13 and 20. In other experiments, cells were cultured in Wnt3aconditioned media or unconditioned media as shown in FIG. 4A (bottom)from days 2-13 or 2-20. G418 selection was imposed on day 7 or 13.Surviving colonies were counted on day 20.

Results showed that Wnt3a conditioned media increases the rate of iPSformation in fibroblasts transduced with the four reprogrammingtranscription factors. As shown in FIG. 4B, Wnt3a promotes iPS cellformation in cells over-exposing Oct4, Sox2, Klf4 and c-Myc. Selectablecells overexpressing Oct4, Sox2, Klf4 and c-Myc formed robustG418-resistant colonies earlier in the presence of Wnt3a conditionedmedia than in the absence of this media. When selected at day 7, onlysmall colonies formed in the absence of Wnt, none of which could bepropagated in culture. Colonies formed in the presence of Wntconditioned media at this point were larger and could be passaged asclones. When selection was started at day 13, colonies were observed inthe absence of Wnt3a conditioned medium that could be propagated.Although there wore fewer colonies at this time point in the presence ofWnt conditioned media than in the absence of Wnt conditioned media, thecolonies that did form were large, relatively homogenous in appearanceand again could be maintained in culture. This result suggests thatWnt3a conditioned medium not only increased the rate of reprogramminghut also selected for colonies of reprogrammed cells.

Wnt3a conditioned media also allows iPS cells to be formed withoutaddition of the oncogenic transcription footer c-Myc. Whereas no iPScells were formed in our initial experiment when fibroblasts weretransduced with Oct4, Sox2 and Klf4, we did observe iPS colonies withthese three factors when cells were grown in Wnt3a conditioned media.These colonies appear to be true iPS cells based on morphology andactivation of the endogenous Oct4 locus, an event normally restricted topluripotent cells. As shown in FIG. 4C, in the presence of Wnt3aconditioned media, robust neo-resistant colonies were observed in Oct4,Sox2, Klf4 overexpressing cells selected at both day 7 and day 13. Inthe absence of Wnt conditioned media, no cells not infected with c-Mycvirus were found to be neo-resistant at either time point Withoutselection, Oct4-IRES-eGFP cells infected with Sox2 and Klf4 lend viruswere found to express GFP (indicative of activation of the endogenousOct4 locus) by day 20 only in the presence of Wnt conditioned media.

Discussion

The findings described above are significant for at least two majorreasons. First, there is great interest m creating iPS cells that do nothave viral integrations of the oncogenic c-Myc transcription factor.Chimaeric mice with iPS cells made with Myc show high rates of cancerassociated with somatic reactivation of the c-Myc virus. Even in vitrowe note that iPS cell lines generated with the c-Myc virus contain amixed population with some cells appearing morphologically much like EScells and others growing more like transformed, cancerous cells. Ourresults obtained thus for indicate that iPS lines created without c-Mycin Wnt3a conditioned media appear to be more homogeneously ES-like intheir morphology. Second, Wnt3a conditioned media appears to exert aselective effect favoring the formation of large, homogenous colonies.Use of Wnt3a conditioned medium or Wnt3a pathway activators could thusbe used as an alternative selection process rather than imposing aselection step that requires genetic modification of the initial somaticcells. Use of Wnt3a conditioned medium or Wnt3a pathway activatorsduring reprogramming would liras provide a valuable improvement to anymethod of reprogramming somatic cells currently known in the art ordeveloped in the future.

Materials and Methods for Examples 2-8

Cell Culture.

V6.5 (C57BL/6-129) murine ES cells and iPS cells were grown undertypical ES conditions on irradiated mouse embryonic fibroblasts (MEFs).Transgenic MEFs used in the infections with DOX-inducible lend viruses(T. Brambrink, R. Foreman, Cell Stem Cell 2, 151-159 (2008)) wereharvested at 13.5 dpc and selected on 2 μg/ml puromycin from embryosafter blastocyst injection of Oct4-IRES-GFPneo/Oct4-inducible ES cells(M. Wernig, A. Meissner, Nature 448, 318-324 (2007)) or harvested fromF1 matings between R26-M2rtTA mice (C. Beard, K. Hochedlinger, Genesis44, 23-28 (2006)) and Oct4-GFP mice (A. Meissner, M. Wernig, NatBiotechnol 25, 1177-1181 (2007). Wnt3a conditioned media and controlconditioned media was generated according to standard protocols (ATCC)(K. Willert, J. D. Brown, Nature 423, 448-452 (2003), described alsoabove) and used in a 1:1 ratio with standard ES cell medium). Wntinhibitor ICG-001 was dissolved in DMSO to a stock concentration of0.1M. The final, working concentration of the Wnt inhibitor was 4 uM.

Viral Transduction.

Tetracycline inducible lentiviral constructs expressing the cDNAs forOct4, Klf4, Sox2 and c-Myc were used as previously described (Brambrink,supra). Virus was prepared by transfecting HBK293T cells with a mixtureof viral plasmid and packaging constructs expressing the viral packagingfunctions and the VSV-G protein (Eugene, Roche). Medium was replaced 24hours after transfection and viral supernatants were collected at 48hours and 72 hours. After filtration, supernatants were pooled and2.5×10⁵ MEFs were incubated with viral supernatants and fresh media at aratio of 1:1 for 24 hours. Infected cells wens then split at ratios from1:5 to 1:12 onto gelatin-coated 10 cm dishes. One day following thesplit, ES media was supplemented with 2 ug/ml DOX and, in theappropriate dishes, conditioned media and/or chemical inhibitor.

Immunostaining and Antibodies Cells were stained as described previously(Wernig, supra). Antibodies against Nanog (Bethyl) and SSEA1 (RADsystems, Minneapolis, Minn.) were used according to supplierrecommendations.

Teratoma Formation

Teratoma formation was assayed as previously described. Briefly, cellswere trypsinized and 5×10⁵ cells were injected subcutaneously into SCIDmice. After 14-21 days, teratomas were dissected, fixed in 10%phosphate-buffered formalin overnight and subsequently embedded inparaffin wax using a Tissue-Tek VIP embedding machine (Miles Scientific,Naperville, Ill.) and a Thermo Shandon Histocenter 2 (Thermo FisherScientific, Waltham, Mass.). Sections were cut at a thickness of 2 μmusing a Leice RM2065 (Leica, Wetzlar, Germany) and stained withhematoxylin and eosin (K. Hochedlinger, Y. Yamada, Cell 121, 465-477(2005).

Blastocyst Injection.

Injections of iPS cells into Balb/c host blastocysts were carried out aspreviously described (Beard, supra).

Example 2: Further Experiments Relating to Generation of ES-Like

To further define the effect of Wnt3a on reprogramming, we infected MEFsthat harbor a doxycycline (DOX)-inducible Oct4 cDNA (Hochedlinger, 2005)with DOX-inducible lentiviral vectors encoding Sox2, Klf4, and c-Myc(Brambrink, et al., 2008). These cells also contained a G418 resistancecassette in the endogenous Oct4 locus allowing for drug selection of iPScells (Meissner et al., 2007).

Four-factor expression was induced by addition of DOX in cells culturedin the presence or absence of Wnt3a conditioned medium (Wnt3a-CM), G418selection was initiated after 5 days and the number of drug resistantcolonies was determined 24 days after induction (FIG. 1a ). FIG. 1bshows that the total number of drug resistant colonies was increasedmore that 7 fold when the cells were cultured in Wnt3a-CM. We also notedthat the drug resistant colonies were larger and more ES-cell like bymorphology when cultured in Wnt3a-CM than in BS cell medium (FIG. 1c ).Furthermore, the colonies that appeared in Wnt3a-CM with G418 selectioninitiated on Day 5 could be further propagated, in contrast to the smallcolonies derived in standard ES cell medium.

Given that Wnt3a-CM had a positive effect on reprogramming in concertwith the four transcription Sectors, we next examined if Wnt3a-CM couldsubstitute for any of the nuclear factors, to parallel experiments,fibroblasts were transduced with subsets of the transcription footersand observed in the presence and absence of Wnt3a-CM (FIGS. 1d and 1e ).No resistant colonies formed in the absence of Oct4 or Klf4 infection.One colony was observed in the absence of Sox2 retrovirus, but thiscolony could not be not be further propagated in ES cell cultureconditions. In contrast, in the presence of Wnt3a-CM, multiple robustG418-resistant colonies formed in the absence of c-Myc in cellsover-expressing Oct4, Sox2 and Klf4 (FIGS. 1d and 1e ). Similar tocolonies from MEFs transduced with all four Actors, these iPS linescould be propagated in standard ES cell media without further selectionand retained ES cell morphology, in replicate experiments,G418-resistant colonies were formed occasionally with no c-Myctransduction in the absence Wnt3a-CM. However, consistent with publishedreports (8,9), these colonies were rare. In the following, iPS cellsgenerated with only three factors and without c-Myc will be designatedas Myc^([−] iPS cells.)

To more closely examine the effects of Wnt3a-CM treatment on thereprogramming process, Oct4/Sox2/Klf4 and Oct4/Sox2/Klf4/c-Mycover-expressing MEFs were cultured with and without and Wnt3a-CM, andG418 selection was initiated at different times after DOX addition. FIG.If shows that when three factor over-expressing cells were cultured inWnt3a-CM medium about 3 fold more Myc^([−]) iPS colonies appeared whenG418 was added at day 5 and about 20 fold more colonies when G418 wasadded at day 10 after induction as compared to cultivation in ES cellmedium (FIG. 1f , left panel) Wnt3a-CM medium also increased the numberof drug resistant colonies after induction of all four factors, thoughthe fold increase was less pronounced than in three factor induced cells(FIG. 1f , right panel). These results indicate that Wnt3a-CM increasedthe number of drug resistant colonies in both three factor and fourfactor induced cells, with the most pronounced effect on three factorover-expressing cells with selection applied at the later time point.

Example 3: Generation of Myc^([−] iPS Clones without Genetic Selection)

Recently, iPS cells have been generated without c-Myc retrovirus(Myc[−]), but in the absence of exogenous c-Myc the efficiency andkinetics of reprogramming are significantly reduced (Nakagawa et al.,2008; Wernig et al., 2008). We tested whether Wnt3a-CM would also aid inthe generation of iPS cells in the absence of selection for Oct4reactivation. For this, cells with GFP driven by the endogenous Oct4promoter were utilized (Meissner, et al., 2008). Oct4/Sox2/Klf4 infectedcells with and without Wnt3a-CM treatment were analyzed for GFPexpression by flow cytometry at days 10, 15 and 20 after DOX induction.No GFP positive cells were present with or without Wnt3a-CM treatment onday 10 or day 15. By day 20 a small population of GFP expressing cellswas detected in cells cultured in Wnt3a-CM but not in standard ES cellmedium (FIG. 1g ). The Wnt3a-CM exposed cultures formed GFP expressingcolonies with morphology typical for ES or iPS cells (FIG. 1b ).However, unlike four footer transduced cells, which usually form ahighly heterogeneous population of cells when propagated withoutselection, the Oct4/Sox2/Klf4/Wnt3a-CM colonies appeared homogenouslyES-like similar to previously reported Myc^([−]) iPS clones (Nakagawa,et al., 2006.

Example 4: Developmental Potential of Myc^([−]) iPS Cells Derived withWnt3a-CM

Several assays were performed to characterize the developmentalpotential of Myc^([−]) iPS cells derived with Wnt3a-CM treatment.Immunocytochemistry confirmed the expression of markers of pluripotency,including the nuclear factor Nanog (FIGS. 2a and 2b ), and the surfaceglycoprotein SSEA1 (FIGS. 2c and 2d ). Functional assays confirmed dial,like ES cells, these iPS cells were pluripotent. When injected into SCIDmice subcutaneously, the Myc^([−]) iPS cells gave rise to teratomas withhistological evidence of cells differentiating into all three germlayers (FIGS. 2e, 2f and 2g ). More importantly, Myc^([−]) iPS cellsderived with Wnt3a-CM treatment contributed to the formation ofdifferentiated tissues in chimeric mice (FIG. 2h ). These resultsindicate that Wnt3a-CM treated Myc^([−]) clones are pluripotent cellsthat are morphologically and functionally indistinguishable from EScells.

Example 5: Effect of Small Molecule Wnt Pathway Inhibitor on Generationof IPS Myc^([−]) and IPS Cells in the Presence of Wnt3a-CM

To quantify the effects of Wnt3a-CM, triplicate experiments wereperformed on Oct4/Sox2/Klf4-inducible, G418 selectable MEFs (FIG. 3a ).G418 was added to the cultures et 15 days after infection to select forcells that had reactivated the Oct4 locus. When scored on day 28 alterinfection, only a few Myc[−] G418 resistant colonies (between 0-3colonies forming on each ten centimetre plate) were detected in standardES cell culture conditions. In contrast, ˜20 fold more drug resistantcolonies formed when G418 selection was initiated on Wnt3a-CM-treatedcells, consistent with the conclusion that activation of the Wnt pathwayenhances reprogramming. It should be noted that conditioned medium fromcontrol fibroblasts lacking Wnt3a over-expression also caused a moderateincrease in the number of G418-resistant colonies relative to standardES medium, suggesting that normal fibroblasts may secrete factors,perhaps including Wnt3a, that promote reprogramming.

To independently assess the effect of Wnt3a on reprogramming, wecultured cells in the presence of ICG-001 (Teo et al., 2005; McMillanand Kahn, 2005; see FIG. 5), an inhibitor of the Wnt/β-catenin pathway.FIG. 3a (right columns) shows that 4 μM ICG-001 strongly inhibited theeffect of Wnt3a-CM on Myc^([−]) iPS formation. The effects of Wnt3a-CMand ICG-001 were also examined in MEFs over-expressing all fourreprogramming factors, including c-Myc (FIG. 3b ). High numbers of G418resistant colonies were observed in both standard ES cell media andWnt3a-CM in four factor reprogrammed cells, with only a subtle increasein the number of colonies with Wnt3aCM. In contrast to the dramaticeffect of ICG-001 on Myc^([−]) cells, at the same dose, the compound hadonly a subtle effect on the number of G418 colonies in c-Myc transducedcells, and a relatively high number of resistant colonies were observedunder these conditions. At higher doses of ICG-001, iPS colony numberswere further reduced, but even at 25 μM multiple Oct4/Sox2/Klf4/c-MyciPS colonies were observed (data not shown). These results areconsistent with the notion that Wnt3a can, at least in part, replace therole of c-Myc in reprogramming.

The Wnt signaling pathway has been shown to connect directly to the coretranscriptional regulatory circuitry of ES cells, suggesting a mechanismby which this pathway could directly promote the induction of thepluripotency in the absence of c-Myc transduction (FIG. 3c ). The Wntsignaling pathway has been shown to connect directly to the cotetranscriptional regulatory circuitry of ES cells, suggesting a mechanismby which this pathway could directly promote the induction of thepluripotency in the absence of c-Myc transduction (FIG. 2c ). In EScells, Tcf3 occupies and regulates the promoters of Oct4, Sox2 and Nanog(Cole et al., 2008; Tam et al., 2008; Yi et al., 2008). In MEFs, theseendogenous pluripotency transcription factors are silenced. Duringreprogramming, as exogenous Oct4, Sox2 and Klf4 contribute to thereactivation of the endogenous pluripotency factors (Jaenisch and Young,2008), Wnt signaling could directly potentiate the effect of thesetranscription factors, as it does in ES cells (Cole et al., 2008).Additionally or alternately, Wnt could serve to activate endogenousc-Myc directly, thereby substituting for exogenous c-Myc. Indeed, c-Mycis a well-established target of the Wnt pathway in colorectal cancercells (He et al., 1098). In ES cells, Tcf3 occupies the c-Myc promoter,and Wnt5a positively contributes to expression of the gene (Cole et al.,2008), The fact that enforced over-expression of c-Myc counteracts thenegative effect of the Wnt inhibitor ICG-001 on the reprogrammingprocess suggests that Wnt stimulation could be acting upstream of theendogenous Myc. Wnt-induced effects on cell proliferation, mediated byc-Myc or other endogenous proliferation factors, could help toaccelerate the sequence of events that lead to the generation of Myc[−]iPS colonies.

A major goal of current research is to identify transient cues that canreprogram somatic cells, eliminating the need for retroviruses. Thestudies described here establish that Wnt stimulation can be used toenhance the efficiency of reprogramming in combination with nuclearActors, Oct4, Sox2 and Klf4. By enhancing the efficiency ofreprogramming in the absence of c-Myc retrovirus, soluble Wnt or smallmolecules that modulate the Wnt signaling pathway will likely proveuseful in combination with other transient cues that can replace theremaining retroviruses.

Example 6: Identification of Additional Reprogramming Agents

Example 3 is modified in that the medium farther contains, in additionto Wnt3a-CM, a candidate reprogramming agent to be tested for itspotential to enhance or inhibit reprogramming. In some embodiments thecells are infected so that they express only 2 of the following 3reprogramming factors: Oct4, Klf4, and Sox2. Agents that enhancegenerating of reprogrammed cells (e.g., increase speed or efficiency ofreprogramming) are identified. The process is repeated to identifyagents capable of substituting for engineered expression of Oct4, Klf4,and/or Sox2 in reprogramming somatic cells.

Example 7: Identification of Additional Reprogramming Agents

Example 3 is modified in that the Wnt3a-CM medium farther contains acandidate reprogramming agent. In some embodiments, the cells areinfected so that they express only 1 or 2 of the following reprogrammingfactors: Oct4, Lin28, Sox2, and Nanog (e.g., Oct4 only, Oct-4 and Sox2).Agents that enhance generating of reprogrammed cells are identified. Theprocess is repeated to identify agents capable of substituting forengineered expression of Oct4, Lin28, Sox2, and/or Nanog inreprogramming somatic cells.

Example 8: Use of Small Molecule Wnt Pathway Modulator in Reprogramming

Example 3 is repeated except that instead of using Wnt3a-CM, ES cellmedium containing a small molecule Wnt pathway activator is used.

REFERENCES

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The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of mouse genetics, developmentalbiology, cell biology, cell culture, molecular biology, transgenicbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art. Such techniques are described in the literature.See, for example, Current Protocols in Cell Biology, ed. by Bonifacino,Dasso, Lippincott-Schwartz, Hartford, and Yamada, John Wiley and Sons,Inc., New York. 1999; Manipulating the Mouse Embryos, A LaboratoryManual, 3^(rd) Ed., by Hogan et al., Cold Spring Contain LaboratoryPress, Cold Spring Contain, New York, 2003; Gene Targeting: A PracticalApproach, IRL Press at Oxford University Press, Oxford, 1993; and GeneTargeting Protocols, Human Press, Totowa, N.J., 2000. All patents,patent applications and references cited herein are incorporated intheir entirety by reference.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and oblate the ends and advantagesmentioned, as well as those inherent therein. The methods, systems andkits are representative of certain embodiments, are exemplary, and arenot intended as limitations on the scope of the invention. Modificationstherein and other uses will occur to those skilled in the art. Thesemodifications are encompassed within the spirit of the invention and aredefined by the scope of the claims. It will be readily apparent to aperson skilled in the art that varying substitutions and modificationsmay be made to the invention disclosed herein without departing from thescope and spirit of the invention.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto Include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more then one, or allof the group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsm which one or more limitations, elements, clauses, descriptive terms,etc., from one or mote of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, e.g., in Markush group orsimilar format, it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should it be understood that, in general, where the invention,or aspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth herein. It shouldalso be understood that any embodiment of the invention can beexplicitly excluded from the claims, regardless of whether the specificexclusion is recited in the specification. For example, any Wntmodulator, e.g., any Wnt pathway activating agent, any somatic celltype, any reprogramming agent, etc., may be excluded.

Where ranges are given herein, the invention includes embodiments inwhich the endpoints are included, embodiments in which both endpointsare excluded, and embodiments in which one endpoint is included and theother is excluded. It should be assumed that both endpoints are includedunless indicated otherwise. Furthermore, it is to be understood thatunless otherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art, values feat areexpressed as ranges can assume any specific value or subrange within thestated ranges in different embodiments of the invention, to the tenth ofthe unit of the lower limit of the range, unless the context clearlydictates otherwise, it is also understood that where a series ofnumerical values is stated herein, the invention includes embodimentsthat relate analogously to any intervening value or range defined by anytwo values in the aeries, and that the lowest value may be taken as aminimum and the greatest value may be taken as a maximum. Numericalvalues, as used herein, include values expressed as percentages. For anyembodiment of the invention in which a numerical value is prefaced by“about” or “approximately”, the invention includes an embodiment inwhich the exact value is netted. For any embodiment of the invention inwhich a numerical value is not prefaced by “about” or “approximately”,the invention includes an embodiment in which the value is prefaced by“about” or “approximately”. “Approximately” or “about” is intended toencompass numbers that fall within a range of ±10% of a number, in someembodiments within ±5% of a number, in some embodiments within ±1%, insome embodiments within ±0.5% of a number, in some embodiments within±0.1% of a number unless otherwise stated or otherwise evident from thecontext (except where such number would impermissibly exceed 100% of apossible value).

Certain claims are presented in dependent form for the sake ofconvenience, but Applicant reserves the right to rewrite any dependentclaim in independent form to include the limitations of the independentclaim and any other claim(s) on which such claim depends, and suchrewritten claim is to be considered equivalent in all respects to thedependent claim in whatever form it is in (either amended or unamended)prior to being rewritten in independent format. It should also beunderstood that, unless clearly indicated to the contrary, in anymethods claimed herein that include more than one act, the order of theacts of the method is not necessarily limited to the older. In which theacts of the method are recited, but the invention includes embodimentsin which the order is so limited.

1.-65. (canceled)
 66. A method of identifying or screening an agentuseful for reprogramming a mammalian somatic cell comprising: (i)identifying an agent that modulates an activity of a Wnt pathway or anagent that substitutes for a reprogramming factor in reprogrammingmammalian somatic cells to a pluripotent state; and (ii) testing theeffect of the agent on reprogramming a mammalian somatic cell.
 67. Themethod of claim 66, wherein the method in step (i) comprises identifyingan agent that modulates an activity of a Wnt pathway.
 68. The method ofclaim 67, wherein the method further comprises in step (i): (a)providing a medium containing the agent; (b) culturing a population ofmammalian somatic cells in the medium; and (c) measuring the activity ofthe Wnt pathway, thereby determining if the agent modulates the activityof the Wnt pathway.
 69. The method of claim 67, further comprises instep (ii) determining, after a period of time, whether cells having oneor more characteristics of pluripotent cells are present aftermaintaining the mammalian somatic cells and their progeny in culture forthe time period.
 70. The method of claim 66, wherein the mammaliansomatic cells are genetically modified or transiently transfected toexpress Oct4, Sox2, and Klf.
 71. The method of claim 66, wherein themethod in step (i) comprises identifying an agent that substitutes for areprogramming factor in reprogramming mammalian somatic cells to apluripotent state.
 72. The method of claim 71, wherein the method instep (i) comprises: (a) introducing into the mammalian somatic cell oneor more reprogramming factors, wherein the reprogramming factor to besubstituted is not introduced into the cell; (b) contacting a populationof mammalian somatic cells with a Wnt pathway activator selected fromthe group consisting of: an exogenous, soluble, biologically active Wntprotein that binds to a Wnt receptor and activates the Wnt pathway, anda small molecule GSK-3 inhibitor that activates the Wnt pathway; (c)contacting the mammalian somatic cells with a candidate agent; (d)maintaining the mammalian somatic cells in a cell culture system for asuitable period of time; and (e) determining whether cells having one ormore characteristics of pluripotent cells are present in said culturesystem, wherein the agent that substitutes for the reprogramming factorin reprogramming mammalian somatic cells is identified if cells havingone or more characteristics of pluripotent cells are present at levelsgreater than would be expected had the mammalian somatic cells not beencontacted with the candidate agent.
 73. The method of claim 72, whereinthe method is for identifying an agent that substitutes for Klf inreprogramming mammalian somatic cells to a pluripotent state, andwherein the method comprises in step (a) introducing into the mammaliansomatic cell reprogramming factors Sox2 and Oct4, but not Klf.
 74. Themethod of claim 72, wherein the method is for identifying an agent thatsubstitutes for Sox2 in reprogramming mammalian somatic cells to apluripotent state, and wherein the method comprises in step (a)introducing into the mammalian somatic cell reprogramming factors Klfand Oct4, but not Sox.
 75. The method of claim 72, wherein the method isfor identifying an agent that substitutes for Oct4 in reprogrammingmammalian somatic cells to a pluripotent state, and wherein the methodcomprises in step (a) introducing into the mammalian somatic cellreprogramming factors Sox2 and Klf, but not Oct4.